Methylbutanol as an advanced biofuel

ABSTRACT

This invention describes genes, metabolic pathways, microbial strains and methods to produce methylbutanol and other compounds of interest from renewable feedstocks.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 12/332,305 filed Dec. 10, 2008, now U.S. Pat. No. 8,114,641; whichclaims the benefit under 35 USC §119(e) to U.S. Application Ser. No.61/012,749 filed Dec. 10, 2007. The disclosure of each of the priorapplications is considered part of and is incorporated by reference inthe disclosure of this application.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention describes genes, polypeptides and expression constructstherefor, metabolic pathways, microbial strains and methods tobiologically produce methylbutanol and derivatives thereof for a varietyof uses including as an advanced biofuel.

2. Background Information

Some oxygenate fuels produced by fermentation, like ethanol, have lowerenergy density than gasoline and absorb water, a property that preventssuch fuels from being distributed with gasoline in existing pipelines.These fuels must be transported separately by rail or trucks to “splash”blending terminals, increasing the cost of blended fuels. Methylbutanol(MBO) has higher energy content than ethanol and because it does notabsorb water, can be distributed on existing pipelines, avoidingadditional transportation costs. Methylbutanol and its derivatives canbe useful as a neat fuel or blend stock for gasoline, diesel, keroseneand jet fuels. Its relatively low volatility also minimizesenvironmental contamination.

SUMMARY OF THE INVENTION

This invention describes genes, polypeptides and expression constructstherefor, metabolic pathways, microbial strains and methods tobiologically produce methylbutanol and derivatives thereof for a varietyof uses including as an advanced biofuel. One embodiment of theinvention relates to a recombinant microorganism comprising at least oneDNA molecule, wherein said at least one DNA molecule encodes at leastthree polypeptides that catalyze a substrate to product conversionselected from the group consisting of: malate to pyruvate, pyruvate tooxaloacetic acid, oxaloacetic acid to L-aspartate, L-aspartate toL-aspartyl-4-phospate, L-aspartyl-4-phospate toL-aspartate-semialdehyde, L-aspartate-semialdehyde to homoserine,homoserine to O-phospho-L-homoserine, and O-phospho-L-homoserine toL-threonine; and wherein said recombinant microorganism produces2-methylbutanol.

In one embodiment, the at least three polypeptides are selected from thegroup consisting of: a) a malate dehydrogenase [EC 1.1.1.37], b) apyruvate carboxylase [EC 6.4.1.1], c) an aspartate aminotransferase [EC2.6.1.1], d) an aspartate kinase [EC 2.7.2.4], e) an aspartic betasemi-aldehyde dehydrogenase [EC 1.2.1.11], f) a homoserine dehydrogenase[EC 1.1.1.3], g) a homoserine kinase [EC 2.7.1.39], and h) a threoninesynthase [EC 4.2.99.2].

The polypeptides used with the invention can be derived from a varietyof sources, such as Picihia, Saccharomyces, or Corynebacterium. Forexample, the malate dehydrogenase may be a Picihia stipitis malatedehydrogenase MDH2 gene product, the pyruvate carboxylase can be aPicihia stipitis pyruvate carboxylase PYC1 gene product, can be a Pichiastipitis aspartate aminotransferase gene AAT2 gene product, and theaspartate kinase can a Pichia stipitis aspartate kinase HOM3 geneproduct. The invention encompasses the use of enzymes, such as HOM3 thathave been modified to become resistant to feedback inhibition.

In another embodiment, the aspartate kinase is derived from the genusSaccharomyces, for example, the aspartate kinase can be a Saccharomycescerevisiae aspartate kinase HOM3 gene product, and further, it can bemodified to become resistant to feedback inhibition. The aspartic betasemi-aldehyde dehydrogenase can be a Pichia stipitis aspartic betasemi-aldehyde dehydrogenase HOM2 gene product, the homoserinedehydrogenase can be a Pichia stipitis homoserine dehydrogenase HOMEgene product, and the homoserine kinase is a Pichia stipitis homoserinekinase THR1 gene product. In still another embodiment, the homoserinekinase can be derived from the genus Corynebacterium, and the threoninesynthase is a Pichia stipitis threonine synthase THR4 gene product.

Another embodiment of the invention relates to a recombinantmicroorganism comprising at least one DNA molecule, wherein said atleast one DNA molecule encodes at least two polypeptides that catalyze asubstrate to product conversion selected from the group consisting of a)L-threonine to 2-oxobutanate, b) 2-oxobutanate to2-aceto-hydroxy-butyrate, c) 2-aceto-hydroxy-butyrate to2,3-dihydroxy-3-methylvalerate, and d) 2,3-dihydroxy-3-methylvalerate to2-keto-3-methylvalerate; and wherein said recombinant microorganismproduces 2-methylbutanol.

In a preferred embodiment, the at least two polypeptides are selectedfrom the group consisting of: a threonine deaminase or a threoninedehydratase [EC 4.3.1.19], an acetolactate synthase [EC 2.2.1.6], or asubunit thereof, a ketol-acid reductoisomerase or an acetohydroxyacidreductoisomerase [EC 1.1.1.86], and a dihydroxyacid dehydratase [EC4.2.1.9]. In one aspect of the invention, the polypeptides are directedto the in the cytoplasm of the recombinant microorganism.

The polypeptides used with the invention can be derived from a varietyof sources, such as Picihia, Escherichia, Saccharomyces, orCorynebacterium.

For example, the threonine deaminase can be a Pichia stipitis threoninedeaminase ILV1 gene product, it can also be a Pichia stipitis threoninedeaminase ILV1 gene product that has been modified to become resistantto feedback inhibition, or modified to optimize cytoplasmic expression.In one embodiment, the threonine deaminase can be derived from the genusSaccharomyces, such as a Saccharomyces cerevisiae ILV1 gene product thathas been modified to become resistant to feedback inhibition. In otherembodiments, the threonine deaminase can be derived from the genusEscherichia or Corynebacterium.

In one aspect of the invention, the acetolactate synthase or anacetolactate synthase subunit is derived from the genus Pichia. Forexample, the acetolactate synthase can be a Pichia stipitis acetolactatesynthase ILV2 gene product, or a Pichia stipitis acetolactate synthaseILV2 gene product that has been modified to optimize cytoplasmicexpression. In another embodiment, the acetolactate synthase is a Pichiastipitis acetolactate synthase ILV6 gene product. In another embodiment,the acetolactate synthase or an acetolactate synthase subunit is derivedfrom the genus Escherichia or Corynebacterium.

In another embodiment of the invention, the ketol-acid reductoisomerasecan be derived from the genus Pichia, Escherichia, or Corynebacterium.For example, the Pichia stipitis ketol-acid reductoisomerase ILV5 geneproduct or an ILV5 gene product that has been modified to optimizecytoplasmic expression.

In another embodiment of the invention, the dihydroxyacid dehydratasederived from the genus Pichia, Escherichia, or Corynebacterium, such asa dihyroxyacid dehydratase is a Pichia stipitis dihydroxyaciddehydratase ILV3 gene product or an ILV3 gene product that has beenmodified to optimize cytoplasmic expression.

Another embodiment of the invention relates to a recombinantmicroorganism comprising at least one DNA molecule, wherein said atleast one DNA molecule encodes (i) a polypeptide that catalyzes a2-keto-3-methyl-valerate to 2-methylbutanal conversion, and (ii) apolypeptide that catalyzes a 2-methylbutanal to 2-methylbutanolconversion; and wherein said recombinant microorganism produces2-methylbutanol. In one aspect, the at least one DNA molecule encodes apyruvate decarboxylase or a pyruvate decarboxylase isoform derived fromthe genus Pichia. For example, the pyruvate decarboxylase can be aPichia stipitis pyruvate decarboxylase PDC3-6 gene product. In anotheraspect, the alpha-ketoacid decarboxylase derived from the genusMycobacterium, the genus Lactococcus. In one embodiment, the alcoholdehydrogenase is derived from the genus Saccharomyces, or the genusPichia. For example, the alcohol dehydrogenase can be a Saccharomycescerevisiae alcohol dehydrogenase ADH6 gene product. In anotherembodiment, the methylglyoxal reductase derived from the genus Pichia orthe genus Saccharomyces.

Another embodiment of the invention relates to a recombinantmicroorganism comprising at least one DNA molecule, wherein said atleast one DNA molecule encodes at least two polypeptides that catalyze asubstrate to product conversion selected from the group consisting of:pyruvate to citramalate, citramalate to erythro-beta-methyl-D-malate,and erythro-beta-methyl-D-malate to 2-oxobutanoate; and wherein saidrecombinant microorganism produces 2-methylbutanol. The polypeptides canbe selected from the group consisting of: a 2-isopropylmalate synthase[EC 2.3.3.13], a citramalate synthase [EC 4.1.3.22], an isopropylmalateisomerase [EC 4.2.1.33], and an isopropylmalate dehydrogenase [EC1.1.1.85].

The polypeptides can be derived from a number of sources, for example,in one embodiment, the 2-isopropylmalate synthase derived from the genusThermatoga or the genus Synechocystis. In one embodiment, thepolypeptide is derived from a Thermotoga maritima 2-isopropylmalatesynthase leuA gene product. In another embodiment, the citramalatesynthase can be derived from the genus Geobacter and the isopropymalateisomerase can be derived from the genus Methanococcus, for example theisopropylmalate isomerase can be a Methanococcus jannaschiiisopropylmalate isomerase leuC gene product and/or leuD gene product.The isopropymalate dehydrogenase can be derived from the genusMethanococcus, such as the Methanococcus jannaschii isopropylmalatedehydrogenase leuB gene product.

Various embodiments of the invention also contemplate a nucleic acidencoding an amino acid biosynthesis regulatory protein, such as aregulatory protein derived from the genus Saccharomyces. In oneembodiment, the regulatory protein can be a Saccharomyces cerevisiaeGCN4 gene product.

Another aspect of the invention relates to methods for producingcompounds. For example, one embodiment of the invention relates to arecombinant method for producing 2-methylbutanol, comprising: providinga culture medium, wherein the culture medium comprises a carbon source;contacting said culture medium with the recombinant microorganism of theinvention, wherein the recombinant microorganism produces spent culturemedium from the culture medium by metabolizing the carbon source to2-methylbutanol; and recovering said 2-methylbutanol from the spentculture medium. In one embodiment, the 2-methylbutanol in the culturemedium is produced at the rate of at least 1500 μM/OD_(60o)/h at 16 hEFT. In another embodiment of the method, the recovering step comprisesextracting 2-methylbutanol using liquid-liquid extraction, wherein asolvent is used to continuously extract at least 2-methylbutanol fromthe spent culture medium. Examples of solvents include diisopropylether, heptane or isooctane. In certain embodiments the solvent isdiisopropyl ether; and at least 90% of 2-methylbutanol is extracted fromthe spent culture medium.

In other embodiments of the invention, the 2-methylbutanol is convertedto bis(2-methyl)butyl ether or 3,3,5-trimethylheptane. In someembodiments, the conversion step of converting 2-methylbutanol tobis(2-methyl)butyl ether comprises treating the 2-methylbutanol with anacid resulting in the formation of bis(2-methyl)butyl ether. An exampleof a suitable acid is trifluoromethanesulfonic acid. In anotherembodiment of the invention, the conversion step of converting2-methylbutanol to bis(2-methyl)butyl ether comprises: refluxing asolution comprising 2-methylbutanol and a catalytic amount of acid;removal of water generated from the solution; and neutralizing thesolution and isolating the ether product.

Examples of products produced include: a compound of formula I, II, III,IV, V or any combination thereof:

In other embodiments of the invention, the conversion step of converting2-methylbutanol is to 3,3,5-trimethylheptane comprises: treating2-methylbutanol with an acid to form 2-methylbutene; hydrogenating2-methylbutene to form 2-methylbutane; and combining 2-methylbutane with2-methylbutene in the presence of acid to form 3,3,5-trimethylheptane.

The disclosed invention also relates to a number of compositionsproduced from biological sources. Preferred embodiments include acomposition comprising a compound of the formula I and/or II:

wherein the compound comprises a fraction of modern carbon (f_(M) ¹⁴C)of at least about 1.003.

In another embodiment, the composition comprising a compound of theformula I, II, III, IV and/or V:

wherein the compound comprises a fraction of modern carbon (f_(M) ¹⁴C)of at least about 1.003.

Embodiments of the invention also include fuel compositions comprising acompound discussed above. Embodiments of these fuel compositions canfurther comprises a petroleum-based fuel, such as gasoline, diesel, jetfuel, kerosene, heating oil, and any combinations thereof. Embodimentsof the fuel composition can further comprises another biofuel. Inanother embodiment, the compounds of the invention can make upapproximately 100% of the composition, or less, such as 1-99% of theweight of the composition or, in another embodiment, 1-99% of the volumeof the composition. In another embodiment, the composition can be a fueladditive or it can comprise a fuel additive.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a metabolic pathway for the preparation of2-methyl-1-butanol. Each enzymatic step of the pathway is provided aletter designation, which corresponds to polypeptides with the followingenzymatic activities: a) malate dehydrogenase; b) pyruvate carboxylase;c) aspartate aminotransferase; d) aspartate kinase; e) aspartic betasemi-aldehyde dehydrogenase; f) homoserine dehydrogenase; g) homoserinekinase; h) threonine synthase; i) threonine deaminase or threoninedehydratase; j) acetolactate synthase; k) ketol-acid reductoisomerase oracetohydroxyacid reductoisomerase; l) dihydroxy-acid dehydratase; m)pyruvate decarboxylase; and n) alcohol dehydrogenase.

FIG. 2 describes an alternative pathway for converting pyruvate tomethylbutanol, wherein steps j) to m) correspond to FIG. 1. Eachenzymatic step of the pathway is provided a letter designation, whichcorresponds to polypeptides with the following enzymatic activities: a′)isopropylmalate synthase or citramalate synthase; b′) isopropylmalateisomerase; and c′) isopropylmalate dehydrogenase.

FIG. 3 depicts a metabolic pathway for the preparation of2-methyl-1-butanol starting from amino acids and other substrates,including leucine, valine, isoleucine, 2-methyl butyric acid,2,3-dihydroxy-3-methyl valeric acid, isoleucine, 2-ketoisovaleric acid,or 2-methylbutyryl CoA. Each enzymatic step of the pathway is provided aletter designation, which corresponds to polypeptides with the followingenzymatic activities: o) or t) valine-isoleucine aminotransferase; u)2-methylbutyrate decarboxylase; v) dihydroxyacid dehydratase; d)2-oxo-acid decarboxylase; e) 2-keto-3-methylvalerate decarboxylase; f)alcohol dehydrogenase; and g) branched chain-alpha-ketoaciddehydrogenase complex.

FIG. 4 depicts a pathway for the preparation of 3-methyl-1-butanolstarting from isovaleryl CoA, leucine, or 2-isopropyl-3-oxosuccinicacid. Each enzymatic step of the pathway is provided a letterdesignation, which corresponds to polypeptides with the followingenzymatic activities: n) ketoisovalerate dehydrogenase complex; o)branched-chain amino acid transaminase; p) leucine aminotransferase; q)leucine dehydrogenase; r) 2-ketoisocaproaste decarboxylase; s)3-methyl-1-butanal reductase; and t) alcohol dehydrogenase.

FIG. 5. Representation of a eukaryotic cell, highlighting the route fromglucose to 2-methylbutanol (2-MBO). The pathway is similar inprokaryotes with the exception that the isoleucine pathway is notconfined to the mitochondria.

FIG. 6. Amino acid analysis of MDH2 (7432), THR1 (7239) and PCK1 (8110)expressed from p415TEF in strain 7123 (ATCC 200869 (MATα ade2Δ::hisGhis3Δ200 leu2Δ0 lys2Δ0 met15Δ0 trp1Δ63 ura3Δ0). Strain 7196 (7123containing empty p415TEF) was included as a control. Significantdifferences in L-threonine content were observed in cultures expressingMDH2 and PCK1 only. Amino acid values are expressed as a percentage ofindividual amino acids in the total amino acid content of the cells.

FIG. 7 shows a bar graph of an amino acid analysis of HOM3 (7245) andHOM3-R2 (7242) expressed from p416TEF in strain 7123. Strain 7123 withempty p416TEF (7209) was included as a control. Significantly lowerL-threonine content was observed in cultures expressing HOM3-R2, whereasno significant difference was observed for wild-type HOM3 and thebackground strain 7209. Amino acid values are expressed as a percentageof individual amino acids in the total amino acid content of the cells.

FIG. 8 shows a bar graph of an amino acid analysis of S. cerevisiae HOM3and HOM3-R2 expressed from p416TEF or p416CYC in strain 7790 (BY4741ΔHOM3::KanMX). Strains are p416TEF-HOM3 (7718), p416TEF-HOM3-R2 (7819),p416CYC-HOM3 (7809) and p416CYC-HOM3-R2 (7805). Cultures were grownovernight in a defined medium lacking threonine and isoleucine to selectfor HOM3 expression. Significantly higher L-threonine content wasobserved in cultures expressing HOM3-R2 from the CYC promoter ascompared to the TEF promoter. A significant increase in L-threoninecontent was also observed when wild-type HOM3 was expressed from TEF ascompared to the CYC promoter. The total L-threonine content of the cellsin the CYC expressed HOM3-R2 culture reached 30% (wt/wt). Amino acidvalues are expressed as a percentage of individual amino acids in thetotal amino acid content of the cells.

FIG. 9 shows a bar graph of an amino acid analysis of S. cerevisiaestrains; 8113 (ΔHOM2) with p416TEF, 8114 p416TEF-PsHOM2, and 7717p416TEF-ScHOM2.

FIG. 10 shows a bar graph of an amino acid analysis of S. cerevisiaestrains 8111 (ΔHOM6) with p415TEF, 8112 p415TEF-PsHOM6, and 7716p415TEF-ScHOM6.

FIG. 11 shows a bar graph of an amino acid analysis of S. cerevisiaestrains 8115 (ΔTHR1) with p415TEF, 8116 p415TEF-PsTHR1, and 7715p415TEF-ScTHR1.

FIG. 12 shows a bar graph of an amino acid composition in BY4741parental strain and KanMX deletions of L-threonine pathway enzymes inthat background. Strains are as follows: 7576=(ΔAAT1), 7577=(ΔAAT2),8177=(ΔMDH1) and 7575=(ΔMDH2). Levels of amino acids were notsignificantly different for any strain other than ΔAAT2, which producedno detectable threonine and significantly higher levels of lysine,glutamate/glutamine and arginine. Amino acid values are expressed as apercentage of individual amino acids in the total amino acid content ofthe cells.

FIG. 13 shows a bar graph of an amino acid analysis of P. stipitis HOM3(8037) and HOM3-R2 (8038) expressed from p416TEF in strain 7790 (BY4741ΔHOM3::KanMX). Cells were grown in defined medium lacking threonine andisoleucine for approximately 24 hrs and extracted as previously stated.Levels of L-threonine were significantly higher in the HOM3-R2 variant,and approached 35% of the total amino acid content of the cells.Although no direct comparison was made, in 7790 the S. cerevisiaeHOM3-R2 variant expressed from CYC promoter achieved 30% threoninecontent compared to 35% for the P. stipitis HOM3-R2 mutant expressedfrom TEF. The wild-type HOM3 from P. stipitis was also found to producemore threonine than wild-type HOM3 from S. cerevisiae (15% vs. 10%) instrain 7790. Amino acid values are expressed as a percentage ofindividual amino acids in the total amino acid content of the cells.

FIG. 14 shows a bar graph of an amino acid analysis of HOM3 (7718) andHOM3-R7 (8118) expressed from p416TEF in strain 7790 (BY4741ΔHOM3::KanMX). Cultures were grown for 48 hrs in a defined mediumlacking threonine and isoleucine to select for HOM3 complementation. Theslow growth of the cells expressing the R7 mutation necessitated theextra 24 hrs of growth compared to cultures expressing the R2 mutation.Significantly higher L-threonine content was observed in culturesexpressing HOM3-R7 from the TEF promoter compared to the native HOM3.The total L-threonine content of the cells in the TEF expressed HOM3-R7culture reached 28% (wt/wt). Amino acid values are expressed as apercentage of individual amino acids in the total amino acid content ofthe cells.

FIG. 15 shows a bar graph of an amino acid analysis of co-expression ofAAT1 (7957), AAT2 (7961), MDH1 (7958), MDH2 (7962) and PCK (7959)together with HOM3-R2. Strain 7960 contained an empty p415TEF plasmid asa control. All enzymes were expressed from p415TEF, with the exceptionof HOM3-R2, which was expressed from p416CYC. Constructs were made instrain 7768 (BY4741 ILV1::TEF-Ilv1-fbr). Cultures were grown for 24 hrsin a defined medium lacking threonine and isoleucine and cells wereprepared for amino acid analysis as described previously. Levels ofamino acids were similar, and threonine content was significantly lowerthan that found in strains containing a wild-type ILV1, indicating thatthreonine is limiting in 2-MBO production. In cells expressing AAT2 andMDH2, higher levels of homoserine were observed, consistent with whatwas observed when HOM3-R2 was expressed by itself. Specifically, the 7%homoserine content found in the culture expressing MDH2 with HOM3-R2 wasthe highest seen in any culture tested, indicating THR1 limitation andpotentially higher flux through the Asp/Thr pathway than HOM3-R2 alone.Also of note is the significantly higher alanine content when PCK isexpressed with HOM3-R2, potentially indicating an increase in pyruvateconcentration, leading to increased alanine. Amino acid values areexpressed as a percentage of individual amino acids in the total aminoacid content of the cells.

FIG. 16 shows a bar graph of an amino acid analysis of P. stipitis HOM3and HOM3-R2 expressed from p416TEF in strain 7790 (HOM3=8039,HOM3-R2=8040) and BY4741 (HOM3=8119, HOM3-R2=8120). Cells were grown indefined medium lacking threonine and isoleucine for approximately 24 hrsand extracted as previously stated. Levels of L-threonine were asobserved before in the 7790 strain (35% total amino acid content), andexpression of this enzyme in the background of chromosomally encoded S.cerevisiae HOM3 was not significantly different. The threonine level forwild-type P. stipitis HOM3 in 7790 was approximately the same aspreviously observed (˜15%). Surprisingly, this same enzyme expressed inthe BY4741 background resulted in a significantly higher threonine levelof approximately 5%, suggesting a contribution from the native HOM3.This could be interpreted as showing a theoretical maximal level ofthreonine achieved by HOM3-R2, since no additional threonine wasdetected by expression in BY4741. Alternatively, it could indicate alimitation in upstream precursors, specifically oxaloacetate, sinceaspartate levels appeared constant in the four different experiments.Additionally, alanine levels showed a significant decrease in theHOM3-R2 strains. One possible explanation is increased pyruvate fluxinto the Asp/Thr pathway via OAA, thereby limiting direct transaminationof pyruvate to alanine. Amino acid values are expressed as a percentageof individual amino acids in the total amino acid content of the cells.

FIG. 17 is a bar graph showing production of 2-MBO by BY4741 deletionvariants transformed with p415TEF-ILV1FBR.

FIG. 18 is a bar graph showing production of 2-MBO by BY4741 deletionvariants transformed with p415TEF-ILV1FBR and p415TEF (empty vectorcontrol).

FIG. 19 is a bar graph showing production of 2-MBO & 3-MBO by BY4741deletion variants transformed with p415TEF-ILV1FBR and p415TEF (emptyvector control).

FIG. 20 is a bar graph with data demonstrating the overexpression ofCHA1 (threonine dehydratase) resulting in an approximately 3 foldincrease in 2-MBO production over the wild-type strain.

FIG. 21 is a bar graph with data demonstrating the overexpression ofILV1 (threonine deaminase) and the feedback resistant (FBR) variantresulting in an approximately 40 fold increase in 2-MBO production overthe wild-type strain. Strain information is provided below:

Strain genotype Strain number BY4741(wild-type) 7766 BY4741 +p415TefIlv1fbr 7746 Ilv1::ilv1fbr 7767 Ilv1::ilv1fbr + p415Tefilv1fbr7747

FIG. 22 (a-e). Data showing the intracellular localization of expressedpolypeptides. The recombinant proteins carry a C-terminal 6× His tag foridentification by the anti-His antibody using immunoblots of specificcell fractions. a) 7541 background ILV1Δ strain with p415TEF ILV1,p415TEF ILV1Δ45 & p415TEF ILV1FBR45; b) 7123 strain with p415TEF ILV2,p415TEF ILV2Δ45; c) 7123 strain with p415TEF ILV5, p415TEF ILV5Δ35; d)7123 strain with p415TEF ILV3, p415TEF ILV3 Δ35, e) 7123 strain withp415TEF ILV3, p415TEF ILV3Δ19 Pichia stipidis (panel ii for ILV3Δ19 is acrude fractionation method showing expression in all 3 fractions). 7.0ug of protein was loaded in each well to all gel results below. A 1:2000dilution of primary antibody was used for each lane. (CR=Crude extract;N=Nuclei fraction; M=Mitochondrial fraction; and Cy=Cytosolic fraction).

FIG. 23 (a-d). Endpoint assays for acetolactate synthase activity werecarried out using pyruvate as substrate. The resulting acetolactate wasconverted to acetone under acidic conditions. Acetone was reacted withα-naphtha for colorimetric detection at 546 nm. E. coli ilvB was foundto the highest activity on pyruvate and E. coli ilvG showed minimumactivity (a). The reaction was only moderately subject to feedbackinhibition by leucine, isoleucine and valine (b, c and d) which could bedue to longer reaction times. Strain information is provided below:

Strain Strain Description number S7209 p416TEF 7209 B p415TEF ilvB (Ec)7302 B + N p415TEF ilvB (Ec) p414TEFilvN (Ec) 8129 G p413TEF ilvG′ (Ec)p415TEF 7307 G + M p415TEF ilvG′ (Ec) p416TEFilvM(Ec) 7558 I p415TEFilvI(Ec) 7560 I + H p415TEF ilvI(Ec) p416TEF ilvH(Ec) 7559 S ce2 p414TEFILV2 p415TEF 7309 2 + 6 p415 TEF ILV6 p414TEF ILV2 7313 B (C glu)p415TEF ilvB (Cg) 7306

FIG. 24 (a-d.) Kinetic curves of keto acid decarboxylases on2-keto-3-methylvalerate a. ΔPDC1; b. L. lactis KdcA; c. Pichia stipitisPDC3-6; d. ES1 (KdcA-PDC1 fusion 1); e. K_(m) values of the four enzymes(a to d) on 2-keto-3-methylvalerate; f. V_(max) values of the fourenzymes (a to d) on 2-keto-3-methylvalerate.

FIGS. 25 (a-f) Kinetic curves of keto acid decarboxylases on 2-ketobutyrate a. APDC1 strain; b. L. lactis KdcA; c. Pichia stipitis PDC3-6;d. ES1 (KdcA-PDC1 fusion 1); e. K_(m) values of the four enzymes (a tod) on 2-keto butyrate; f. V_(max) values of the four enzymes (a to d) on2-keto butyrate.

FIG. 26 (a-e). Kinetic curves of keto acid decarboxylases on pyruvate a.ΔPDC1; b. L. lactis KdcA; c. Pichia stipitis PDC3-6; d. ES1 (KDCA-PDC1fusion 1); e. V_(max) values of the four enzymes (a to d) on pyruvate.

FIG. 27. Activity of S. cerevisiae ADH1 on various aldehydes.

FIG. 28 (a and b). Kinetic curves of ADH6 on acetaldehyde (a) and2-methylbutyraldehyde (b).

FIG. 29 (a and b). Km and Vmax values of S. cerevisiae ADH6 on thesubstrates acetaldehyde and 2-methylbutyraldehyde.

FIG. 30. Co-factor oxidation of GRE2 (alcohol dehydrogenase) onmethylbutyraldhyde substrates.

FIG. 31. Co-factor oxidation of decarboxylase and alcohol dehydrogenasecombinations. Strains 7632 (p416TEF-ADH6, p415TEF-PDC1); 7633(p416TEF-His:GRE2), pTEF-PDC5); 7634 (ΔPDC1, p416TEF-ADH6,p415TEF-PDC1); 7635 (p416TEF-His:GRE2, p415TEF-PDC1); 7636(p416TEF-ADH6, p415TEF-KdcA(L1)); 7637 (p416TEF-ADH6,p415TEF-KdcA(L1)-5286Y); 7638 (p416TEF-ADH6, p415TEF-KdcA(Mt); 7639(p416TEF-ADH6, p415TEF-PDC5); 7640 (p416TEF-His:GRE2, p415TEF-KdcA(L1));7641 (p416TEF-His:GRE2, p415TEF-KdcA(L1)-S286Y).

FIG. 32. Alignment of KdcAp and Pdclp from Lactococcus lactis andSaccharomyces cerevisiae, respectively. Amino acids identified for sitesaturation mutagenesis are outlined by the boxes.

FIG. 33. Schematic for making mutant library. Amplification 1 and 2 werecompleted separately via PCR then combined in one PCR round to attainAmplification 3. Amplification 3 could have both mutations as well assingle mutations. These mutations could also be stop codons.

FIG. 34. Schematic of KdcAp structure. The boxed regions are those thatwere fusion sites when combining KdcAp to Pdc1p (Exchange Sites).

FIG. 35. Schematic of Pdc1p structure. The boxed regions are those thatwere fusion sites when combining KdcAp to Pdc1p (Exchange Sites).

FIG. 36. Schematic of the KdcAp and Pdc1p fusion protein at ExchangeSite #1 as indicated in FIG. 35. This Exchange Site is in a conservedregion for both proteins in domain 1.

FIG. 37. Schematic of the KdcAp and Pdc1p fusion protein at ExchangeSite #2 as indicated in FIG. 35. This Exchange Site is in a conservedregion for both proteins in between domain 2 and domain 3.

FIG. 38. Schematic of the KdcAp and Pdc1p fusion protein at ExchangeSite 3. This Exchange Site is in a conserved region for both proteins inbetween domain 2 and domain 3.

FIG. 39. Diagrammatic representation of strategy for creating fusionproteins between Lactococcus lactis KDCA and Pichia stipitis PDC3-6 toincrease the affinity for branched keto acids.

FIG. 40. A schematic of KdcA-PDC3-6 fusion 1 for increasing the affinityfor brance keto acids.

FIG. 41. A schematic of KdcA-PDC3-6 fusion 2 for increasing the affinityfor brance keto acids.

FIG. 42. A schematic of PCR amplification of cassettes. Primers give 40base overlaps between primary cassettes.

FIG. 43. A schematic of amplification of Truncated-Hybrid Cassettes fromOverlapping Primary Cassettes.

FIG. 44. A schematic of fragments used in an isothermal reaction thatcreates 7 kb fragment. Note there are 40 base overlaps between thefragments.

FIG. 45. A schematic of fragments used in an isothermal reaction creates8 kb fragment. Note there are 40 base overlaps between the fragments.

FIG. 46. A schematic of fragments used in an isothermal reaction orIn-vivo recombination by Yeast. Note there are 40 base overlaps betweenthe fragments. These are used for both isothermal assembly and In-vivorecombination by yeast.

FIG. 47. A graph of 2-MBO production during fermentation of strainsexpressing threonine deaminase (TD). Fermentation were performed understandard conditions (aerobic, 30° C., pH 4.5) in YNB medium containing50 g 1-1 glucose and supplemented with nutrients to complement remainingauxotrophies.

FIG. 48. A graph of specific 2-MBO production in strains expressing TDand AK. Fermentations were preformed under standard conditions (aerobic,30° C., pH4.5). Specific productivity was calculated from interpolatedvalues for biomass and 2-MBO. The average of 2 independent replicatesare reported.

FIG. 49. A graph of specific productivity of 2-MBO with excess 2-MBA.Fermentations were performed under standard conditions (aerobic, 30° C.,pH 4.5) in YNB medium containing 50 g 1-1 glucose and supplemented withnutrients to complement auxotrophies. A bolus of 2-MBA was added andsamples collects hourly thereafter. Specific productivity was calculatedfrom interpolated values for 2-MBO and biomass.

FIG. 50. A graph of 2-MBO production by strain 7338 (p415TEF ILV1FBR) inmedium with increased glucose. Fermentations were performed understandard conditions (aerobic, 30° C., pH 4.5) in YNB medium containingthe indicated amount of glucose. In one set of experiments, a constantglucose feed was employed. The data shown represent the average of twoindependent replicates.

FIG. 51. A graph of 2-MBO specific productivity in strains expressingGCN4. Fermentations were performed under standard conditions (aerobic,30° C., pH 4.5) in YNB containing 50 g 1-1 glucose and supplemented withnutrients to complement auxotrophies.

FIG. 52. The heterologous citramalate pathway composed of cimA/leuA,leuB, leuC, and leuD is expressed in the cytoplasm of S. cerevisiae.

FIG. 53. A graph showing results of detection of citramalate via HPLC.

FIG. 54. A bar graph showing MBO production of cimA/leuA 1 clones. A 3-to 8-fold increase in 2-MBO was observed for strains containingcimA/leuA, leuB, and leuCD and grown in +Ile SD medium. Straininformation shown in Table 10.

FIG. 55. A bar graph showing citramalate production of variousheterologous genes in Saccharomyces. Strains 8123 control strain (emptyvector); 8055 p416GPD-cimA (Tm)+p415TEF1; 8059 p416GPD-cimA (Gs),p415TEF1.

FIG. 56. A bar graph showing calculated maximum theoretical yields of2-MBO from glucose in yeast resulting from different geneticmanipulations.

FIG. 57. Effect of Isoleucine on the putative leuA activity andvariants. Isoleucine was added to 10 mM.

FIG. 58 (a-c). Shows tracings of GCMS analysis of MBO production.

FIG. 59. Shows a map of YAC6 comprising the following genes: ilvAfbr(Ec), ilvG′ (Ec), ilvC (Ec), ilvD (Ec), kdcA (L1), adh6.

FIG. 60. Shows a map of YAC8 comprising the following genes: mdh2 (Ps),pyc1 (Ps), aat2 (Ps), hom3fbr (Ps), hom2 (Ps), hom6 (Ps), thr1 (Ps),thr4 (Ps).

FIG. 61. Shows a map of YAC10 comprising the following genes: mdh2 (Ps),pyc1 (Ps), aat2 (Ps), hom3fbr (Ps), hom2 (Ps), hom6 (Ps), thr1 (Ps),thr4 (Ps), kdcA (L1), adh6.

FIG. 62. Shows a map of YAC9 comprising the following genes: hom3fbr(Ps), pyc1 (Ps), aat2 (Ps), hom3fbr (Ps), hom2 (Ps), hom6 (Ps), thr1(Ps), thr4 (Ps), pdc3-6 (Ps), adh6.

FIG. 63. Shows a map of YAC9 with hom3fbr on CUP promoter comprising thefollowing genes: hom3fbr (Ps), pyc1 (Ps), aat2 (Ps), hom3fbr (Ps), hom2(Ps), hom6 (Ps), thr1 (Ps), thr4 (Ps), pdc3-6 (Ps), adh6.

FIG. 64. Shows a map of YAC7 comprising the following genes: hom3fbr(Ps), pyc1 (Ps), aat2 (Ps), hom3fbr (Ps), hom2 (Ps), hom6 (Ps), thr1(Ps), thr4 (Ps).

FIG. 65. Shows a map of YAC7 with hom3fbr on CUP promoter comprising thefollowing genes: hom3fbr (Ps), pyc1 (Ps), aat2 (Ps), hom3fbr (Ps), hom2(Ps), hom6 (Ps), thr1 (Ps), thr4 (Ps).

FIG. 66. Shows a map of YAC5 comprising the following genes: ilv1fbr(Ps), ilv2 (Ps), ilv6 (Ps), ilv5 (Ps), ilv3 (Ps).

FIG. 67. Shows a map of YAC5 with ilv1fbr on CUP promoter comprising thefollowing genes: ilv1fbr (Ps), ilv2 (Ps), ilv6 (Ps), ilv5 (Ps), ilv3(Ps).

FIG. 68. Shows a map of YAC5 truncated comprising the following genes:ilv1fbrΔ15 (Ps), ilv2Δ26 (Ps), ilv6 (Ps), ilv5Δ40 (Ps), ilv3Δ34 (Ps).

FIG. 69. Shows a map of YAC5 truncated with ilv1fbr on CUP promoterilv1fbrΔ15 (Ps), ilv2Δ26 (Ps), ilv6 (Ps), ilv5Δ40 (Ps), ilv3Δ34 (Ps).

FIG. 70. Shows a map of YAC14 comprising the following genes: hom3fbr(Ps), pyc1 (Ps), aat2 (Ps), hom3fbr (Ps), hom2 (Ps), hom6 (Ps), thr1(Ps), thr4 (Ps), pdc3-6 (Ps), adh6, ilv1fbr (Ps), ilv2 (Ps), ilv6 (Ps),ilv5 (Ps), ilv3 (Ps).

FIG. 71. Shows a map of YAC14 with hom3fbr, ilv1fbr on CUP promotercomprising the following genes: hom3fbr (Ps), pyc1 (Ps), aat2 (Ps),hom3fbr (Ps), hom2 (Ps), hom6 (Ps), thr1 (Ps), thr4 (Ps), pdc3-6 (Ps),adh6, ilv1fbr (Ps), ilv2 (Ps), ilv6 (Ps), ilv5 (Ps), ilv3 (Ps).

FIG. 72. Shows a map of YAC14 with ILV truncations comprising thefollowing genes: hom3fbr (Ps), pyc1 (Ps), aat2 (Ps), hom3fbr (Ps), hom2(Ps), hom6 (Ps), thr1 (Ps), thr4 (Ps), pdc3-6 (Ps), adh6, ilv1fbrΔ15(Ps), ilv2Δ26 (Ps), ilv6 (Ps), ilv5Δ40 (Ps), ilv3Δ34 (Ps).

FIG. 73. Shows a map of YAC14 with ILV truncations, hom3fbr, ilv1fbr onCUP promoter comprising the following genes: hom3fbr (Ps), pyc1 (Ps),aat2 (Ps), hom3fbr (Ps), hom2 (Ps), hom6 (Ps), thr1 (Ps), thr4 (Ps),pdc3-6 (Ps), adh6, ilv1fbrΔ15 (Ps), ilv2Δ26 (Ps), ilv6 (Ps), ilv5Δ40(Ps), ilv3Δ34 (Ps).

FIG. 74 (a-c). a) Shows a representative chromatogram for the array ofC1-C4 alcohols, 2-MBO and isovaleric acid. The internal standard1-pentanol. b) Shows a representative chromatogram for the alcohols andMBOs in a fermentation broth sample. The internal standard 1-pentanol.Since the relative response factor for 3-MBO is similar to that for2-MBO, the quantification of 3-MBO is based on the calibration curve setfor 2-MBO. c) Shows a representative chromatogram: 1-Methanol;2-Ethanol; 3-n-Propanol; 4-iso-Butanol; 5-n-Butanol; 6-2 MBO (partialseparation from 3 MBO); 7-2MeBu Acid (co-elution with isovaleric acid).

FIG. 75 (a-c). Shows representative chromatograms. a) Short run, afterimproving resolution (Rtx-624 20×0.18, 1 um, MBOorg2), b) Short run(Rtx-624 20×0.18, 1 um, MBOorg1), and c) long run (Rtx-624 30×0.25, 1.4um, MBOFASTC/GC1).

DETAILED DESCRIPTION OF THE INVENTION

The invention described herein relates to recombinant microorganismscapable of metabolizing a variety of carbon sources to a number ofcommercially valuable compounds, including isoamyl alcohol, propanol,methylbutanols (MBO) such as 2-methyl-1-butanol (2-MBO),3-methyl-1-butanol (3-MBO), and isobutanol. Derivatives of thesecompounds are also contemplated, and may be synthesized eitherbiologically or chemically. For example, derivatives of methylbutanolinclude 2-methyl-1-(2-methylbutoxy)butane and1-(isopentyloxy)-3-methylbutane, 1-(isopentyloxy)-2-methylbutane,2-methyl-1-(tert-pentyloxy)butane, and2-methyl-2-(tert-pentyloxy)butane. The recombinant microorganisms areengineered to include a variety of heterologous genes encoding enzymeswhich complement or replace endogenous enzymatic systems. The inventionalso describes fuel compositions containing the compounds produced bythe recombinant microorganisms and derivatives thereof, as well asmethods of using such compositions.

FIG. 1 shows a proposed pathway for the generation of MBO with malate asthe starting material. Each step of the enzymatic pathway is providedwith a letter designation which corresponds to an polypeptide with thefollowing enzymatic activity.

-   -   Step a) corresponds to the conversion of malic acid to pyruvic        acid,    -   Step b) corresponds to the conversion of pyruvic acid to        oxaloacetic acid,    -   Step c) corresponds to the conversion of oxaloacetic acid to        L-aspartic acid,    -   Step d) corresponds to the conversion of L-aspartic acid to        L-aspartyl-4-phospate,    -   Step e) corresponds to the conversion of L-aspartyl-4-phospate        to 2-amino-4-oxo-butanoic acid (L-aspartate semialdehyde),        step f) corresponds to the conversion of 2-amino-4-oxo-butanoic        acid (L-aspartate semialdehyde) to homoserine,    -   Step g) corresponds to the conversion of homoserine to        O-phospho-L-homoserine,    -   Step h) corresponds to the conversion of O-phospho-L-homoserine        to L-threonine,    -   Step i) corresponds to the conversion of L-threonine to        2-oxobutanic acid,    -   Step j) corresponds to the conversion of 2-oxobutanic acid to        2-aceto-hydroxy-butyric acid,    -   Step k) corresponds to the conversion of 2-aceto-hydroxy-butyric        acid to 2,3-dihydroxy-3-methylvaleric acid,    -   Step l) corresponds to the conversion of        2,3-dihydroxy-3-methylvaleric acid to 2-keto-3-methylvaleric        acid,    -   Step m) corresponds to the conversion of 2-keto-3-methylvaleric        acid to 2-methylbutanal, and    -   Step n) corresponds to the conversion of 2-methylbutanal to        2-methylbutanol.

The designations provide examples of enzymatic activities that catalyzeparticular reactions in the overall pathway. For example, malatedehydrogenase is an example of a designation for the enzyme thatcatalyzes the conversion of malate to pyruvate. Because enzymaticnomenclature various between organisms, it should be noted that thenames provided above are merely illustrative of a class of enzymes thatcatalyze the particular steps of the pathway. The enzymes contemplatedfor use with the invention are those that catalyze the reactionsillustrated and are not limited to the enzymatic names provided.

Polypeptides providing the following enzymatic activities correspondingto the steps of FIG. 1 are:

-   -   Step a) malate dehydrogenase [EC 1.1.1.37]    -   Step b) pyruvate carboxylase [EC 6.4.1.1]    -   Step c) aspartate aminotransferase [EC 2.6.1.1];    -   Step d) aspartate kinase or L-aspartate-4-P-transferase [EC        2.7.2.4];    -   Step e) aspartic beta semi-aldehyde dehydrogenase [EC 1.2.1.11];    -   Step f) homoserine dehydrogenase [EC 1.1.1.3];    -   Step g) homoserine kinase [EC 2.7.1.39];    -   Step h) threonine synthase [EC 4.2.99.2];    -   Step i) threonine deaminase or threonine dehydratase [EC        4.3.1.19];    -   Step j) acetolactate synthase or a subunit thereof [EC 2.2.1.6];    -   Step k) ketol-acid reductoisomerase or acetohydroxyacid        reductoisomerase [EC 1.1.1.86];    -   Step l) dihydroxy-acid dehydratase [EC 4.2.1.9];    -   Step m) pyruvate decarboxylase [EC 4.1.1.1] or alpha-keto acid        decarboxylase [4.1.1.72]; and    -   Step n) alcohol dehydrogenase [EC 1.1.1.1]. The EC numbers        provided use the enzyme nomenclature of the Nomenclature        Committee of the International Union of Biochemistry and        Molecular Biology.

A first aspect of the invention provides a recombinant microorganismcomprising at least one DNA molecule, wherein said at least one DNAmolecule encodes at least three polypeptides that catalyze a substrateto product conversion selected from the group consisting of steps a)through h), wherein said recombinant microorganism produces2-methylbutanol.

In particular embodiments of the invention, the polypeptide catalyzingthe conversion of malate to pyruvate is derived from a yeast. An exampleof a suitable source for this enzyme is the genus Pichia, a preferredsource is Picihia stipitis.

A specific example of a suitable sequence is:

Pichia stipitis MDH2 (Ps) amino acid sequence:

(SEQ ID NO: 1) MPHSVTPSIEQDSLKIAILGAAGGIGQSLSLLLKAQLQYQLKESNRSVTHIHLALYDVNQEAINGVTADLSHIDTPISVSSHSPAGGIENCLHNASIVVIPAGVPRKPGMTRDDLFNVNAGIISQLGDSIAECCDLSKVFVLVISNPVNSLVPVMVSNILKNHPQSRNSGIERRIMGVTKLDIVRASTFLREINIESGLTPRVNSMPDVPVIGGHSGETIIPLFSQSNFLSRLNEDQLKYLIHRVQYGGDEVVKAKNGKGSATLSMAHAGYKCVVQFVSLLLGNIEQIHGTYYVPLKDANNFPIAPGADQLLPLVDGADYFAIPLTITTKGVSYVDYDIVNRMNDMERNQMLPICVSQLKKNIDKGLEFVASRSASS.

Another exemplary sequence is:

Saccharomyces cerevisiae MDH2 amino acid sequence:

(SEQ ID NO: 2) MVKVTVCGAAGGIGQPLSLLLKLNPAVSELALFDIVNAKGVAADLSHINTPAVVTGHQPANKEDKTAIVDALKGTDLVVIPAGVPRKPGMTRADLFNINASIIRDLVANIGRTAPNAAILIISNPVNATVPIAAEVLKKLGVFNPGKLFGVTTLDSVRAETFLGELINVNPSQLQGRISVVGGHSGDTIVPLINVTPDVSAKVANISKADYDKFVNRVQFGGDEVVKAKNGAGSATLSMAYAGYRFAAGVLNSLGGASTSSSGVPDSSYVYLPGVPGGKEFSAKYLNGVDFFSVPIVLENGVIKSFINPFEHMKITQKEQELVKVALGGLEKSIEQGTNF VKGSKL

In particular embodiments of the invention, the polypeptide catalyzingthe conversion of pyruvate to oxaloacetic acid is derived from a yeast.An example of a suitable source for this enzyme is the genus Pichia, apreferred source is Picihia stipitis.

A specific example of a suitable sequence is:

Pichia stipitis PYC1 (Ps) amino acid sequence:

(SEQ ID NO: 3) MSSLSPHDHHGKINQMRRDSTVLGPMNKILVANRGEIPIRIFRTAHELSMQTVAIYSHEDRLSMHRLKADESYVIGKKGEFSPVGAYLQIDEIIKIAKTHNVNMIHPGYGFLSENSEFARKVEEAGIAWIGPTHETIDAVGDKVSARNLALANDVPVVPGTPGPIDSVEEAEAFVEKYGYPVIIKAAFGGGGRGMRVVREGDDIGDAFKRATSEAKTAFGNGTCFIERFLDKPKHIEVQLLADGYGNVIHLFERDCSVQRRHQKVVEIAPAKNLPKAVRDAILTDAVKLAKSANYRNAGTAEFLVDEQNRHYFIEINPRIQVEHTITEEITGVDIVAAQIQIAAGASLQQLGLLQDKITTRGFAIQCRITTEDPSKNFQPDTGKIEVYRSSGGNGVRLDGGNGFAGSIISPHYDSMLVKCSTSGSTYEIARRKMLRALIEFRIRGVKTNIPFLLALLTNETFISGSCWTTFIDDTPSLFQMISSQNRANKILSYLADLIVNGSSIKGQVGLPKLNEEAEIPTIHDPKTGIPIDVELNPAPRGWRQVLLEEGPDAFAKKVRNFNGTLITDTTWRDAHQSLLATRLRTIDLLNIAPTTAHALNGAFSLECWGGATFDVCMRFLYEDPWARLRKLRKLVPNIPFQMLLRGANGVAYSSLPDNAIDQFVKQAKDNGVDIFRVFDALNDLDQLKVGIDAVKKAGGVVEATVCYSGDMLQKGKKYNLAYYVDVVDKIVAMGTHFLGIKDMAGTLKPKAATDLVSAIRAKYPDLPIHVHTHDSAGTGVASMTAAAKAGADVVDAASNSMSGMTSQPSISAILASFEGEVETGLSERLVREIDHYWAQMRLLYSCFEADLKGPDPEVYEHEIPGGQLTNLLFQAQQLGLGAKWLQTKETYKIANRVLGDVVKVTPTSKVVGDLAQFMVSNNLTEEDVNKLAGELDFPDSVLDFMEGLMGTPYGGFPEPLRTNMLGNKRQKLNERPGLSLAPVDFSALKQELVSKYGNSIKEVDLASYTMYPKVYESYRKIVEKYGDLSVLPTRYFLKGINVGEELSVEIEQGKTLIVKLLAVGEISQQKGTREVFFELNGEMRSVTVDDKTVSVETITRRKATQPNEVGAPMAGVVIEIRTQSGTDVKKGDPIAVLSAMKMEMVISAPVSGVVGEILIKEGESVDASDLITSILKHN.

Other exemplary sequences are:

Saccharomyces cerevisiae PYC1 amino acid sequence:

(SEQ ID NO: 4) MSQRKFAGLRDNFNLLGEKNKILVANRGEIPIRIFRTAHELSMQTVAIYSHEDRLSTHKQKADEAYVIGEVGQYTPVGAYLAIDEIISIAQKHQVDFIHPGYGFLSENSEFADKVVKAGITWIGPPAEVIDSVGDKVSARNLAAKANVPTVPGTPGPIETVEEALDFVNEYGYPVIIKAAFGGGGRGMRVVREGDDVADAFQRATSEARTAFGNGTCFVERFLDKPKHIEVQLLADNHGNVVHLFERDCSVQRRHQKVVEVAPAKTLPREVRDAILTDAVKLAKECGYRNAGTAEFLVDNQNRHYFIEINPRIQVEHTITEEITGIDIVAAQIQIAAGASLPQLGLFQDKITTRGFAIQCRITTEDPAKNFQPDTGRIEVYRSAGGNGVRLDGGNAYAGTIISPHYDSMLVKCSCSGSTYEIVRRKMIRALIEFRIRGVKTNIPFLLTLLTNPVFIEGTYWTTFIDDTPQLFQMVSSQNRAQKLLHYLADVAVNGSSIKGQIGLPKLKSNPSVPHLHDAQGNVINVTKSAPPSGWRQVLLEKGPAEFARQVRQFNGTLLMDTTWRDAHQSLLATRVRTHDLATIAPTTAHALAGRFALECWGGATFDVAMRFLHEDPWERLRKLRSLVPNIPFQMLLRGANGVAYSSLPDNAIDHFVKQAKDNGVDIFRVFDALNDLEQLKVGVDAVKKAGGVVEATVCFSGDMLQPGKKYNLDYYLEIAEKIVQMGTHILGIKDMAGTMKPAAAKLLIGSLRAKYPDLPIHVHTHDSAGTAVASMTACALAGADVVDVAINSMSGLTSQPSINALLASLEGNIDTGINVEHVRELDAYWAEMRLLYSCFEADLKGPDPEVYQHEIPGGQLTNLLFQAQQLGLGEQWAETKRAYREANYLLGDIVKVTPTSKVVGDLAQFMVSNKLTSDDVRRLANSLDFPDSVMDFFEGLIGQPYGGFPEPFRSDVLRNKRRKLTCRPGLELEPFDLEKIREDLQNRFGDVDECDVASYNMYPRVYEDFQKMRETYGDLSVLPTRSFLSPLETDEEIEVVIEQGKTLIIKLQAVGDLNKKTGEREVYFDLNGEMRKIRVADRSQKVETVTKSKADMHDPLHIGAPMAGVIVEVKVHKGSLIKKGQPVAVLSAMKMEMIISSPSDGQVKEVFVSDGENVDSSDLLVLLEDQVPVETKASaccharomyces cerevisiae PYC2 amino acid sequence:

(SEQ ID NO: 5) MSSSKKLAGLRDNFSLLGEKNKILVANRGEIPIRIFRSAHELSMRTIAIYSHEDRLSMHRLKADEAYVIGEEGQYTPVGAYLAMDEIIEIAKKHKVDFIHPGYGFLSENSEFADKVVKAGITWIGPPAEVIDSVGDKVSARHLAARANVPTVPGTPGPIETVQEALDFVNEYGYPVIIKAAFGGGGRGMRVVREGDDVADAFQRATSEARTAFGNGTCFVERFLDKPKHIEVQLLADNHGNVVHLFERDCSVQRRHQKVVEVAPAKTLPREVRDAILTDAVKLAKVCGYRNAGTAEFLVDNQNRHYFIEINPRIQVEHTITEEITGIDIVSAQIQIAAGATLTQLGLLQDKITTRGFSIQCRITTEDPSKNFQPDTGRLEVYRSAGGNGVRLDGGNAYAGATISPHYDSMLVKCSCSGSTYEIVRRKMIRALIEFRIRGVKTNIPFLLTLLTNPVFIEGTYWTTFIDDTPQLFQMVSSQNRAQKLLHYLADLAVNGSSIKGQIGLPKLKSNPSVPHLHDAQGNVINVTKSAPPSGWRQVLLEKGPSEFAKQVRQFNGTLLMDTTWRDAHQSLLATRVRTHDLATIAPTTAHALAGAFALECWGGATFDVAMRFLHEDPWERLRKLRSLVPNIPFQMLLRGANGVAYSSLPDNAIDHFVKQAKDNGVDIFRVFDALNDLEQLKVGVNAVKKAGGVVEATVCYSGDMLQPGKKYNLDYYLEVVEKIVQMGTHILGIKDMAGTMKPAAAKLLIGSLRTRYPDLPIHVHSHDSAGTAVASMTACALAGADVVDVAINSMSGLTSQPSINALLASLEGNIDTGINVEHVRELDAYWAEMRLLYSCFEADLKGPDPEVYQHEIPGGQLTNLLFQAQQLGLGEQWAETKRAYREANYLLGDIVKVTPTSKVVGDLAQFMVSNKLTSDDIRRLANSLDFPDSVMDFFEGLIGQPYGGFPEPLRSDVLRNKRRKLTCRPGLELEPFDLEKIREDLQNRFGDIDECDVASYNMYPRVYEDFQKIRETYGDLSVLPTKNFLAPAEPDEEIEVTIEQGKTLIIKLQAVGDLNKKTGQREVYFELNGELRKIRVADKSQNIQSVAKPKADVHDTHQIGAPMAGVIIEVKVHKGSLVKKGESIAVLSAMKMEMVVSSPADGQVKDVFIKDGESVDASDLLVVLEEETLPPSQKKPichia stipitis PYC2 (Ps) amino acid sequence:

(SEQ ID NO: 6) MTASSLDNQLNYVHAAFDEENDGLLPISLQDLTNKHKEASTSKNSTFAPKNTSLPSSTKSASLLKVDRPAFFVLVLLYLLQGVPVGLAFGSIPFILKSKLSYSQVGIFSLAAYPYSLKLIWSPIVDAVYSPKLGRRRSWIIPIQTISGVTLIYLGSLIDGLMEDPQNCLPTITFCFFMLVFFCATQDIAVDGWALTCLSPESLSYASTAQTIGINTGYFSSFTIFLALSSPDFANRYLRKVPLDVGLFSLGSYLTFWGWMFLAVTALLWFVPEDPPHLAKRNQAKLSNEKIKTESVYNKDSKFKDLQNVYLAMFKVLKLPNVQTFVIILLISKFGFQVNEAATNLKLLEKGLSKEDLSITVLIDFPFEMVFGYYAGRWSTGKSPLKPWIFGFAGRLVAAALAQGIVYFFPEDGKISSFYFLLVILQHLLGSFMSTIQFVSLCAFHTKIADPAIGGTYMTTLNTLSNYGGTWPRLILLYLIDKLTIEECKVPSVTNSYYITDEDLRQQCKSSGGKLTVLRDGYYYTNTICVIIGIFTLLWVKRKTTYLQSL PNSAWRVNKD

In particular embodiments of the invention, the polypeptide catalyzingthe conversion of oxaloacetic acid to L-aspartate is derived from ayeast. An example of a suitable source for this enzyme is the genusPichia, a preferred source is Picihia stipitis.

A specific example of a suitable sequence is:

Pichia stipitis AAT2 (Ps) amino acid sequence:

(SEQ ID NO: 7) MSYFAGITELPPDPLFGLKARYVADSRTDKVDLGIGAYRDNNGKPWILPAVKLAEAKLVSSPDYNHEYLSISGFEPFLKQASKVILGENSAALAENRVVSQQSLSGTGALHVAGVLLKEFYTGEKTVYLSKPTWANHNQIFTSIGFKVASYPYWDNDTKSLDLKGFLSTIRTAPAGSIFLLHACAHNPTGLDPSQDEWKQVLKELEAKKHLVLFDSAYQGFASGDLDKDAYAIRYAIDQKVISTPIIICQSFAKNVGMYGERVGAIHVIPSTQKDEQLGRALKSQLNRIIRSEISNPPAYGAKIVSTILNDRALRQQWEADLVTMSSRIHKMRLKLKELLTNLHTPGTWDHIVNQTGMFSFTGLSPDMVARLEKVHGIYLVSSGRASVAGLNDGNVEKVA NAIDEVVRFYAKPKL

Other exemplary sequences are:

Saccharomyces cerevisiae AAT1 amino acid sequence:

(SEQ ID NO: 8) MLRTRLTNCSLWRPYYTSSLSRVPRAPPDKVLGLSEHFKKVKNVNKIDLTVGIYKDGWGKVTTFPSVAKAQKLIESHLELNKNLSYLPITGSKEFQENVMKFLFKESCPQFGPFYLAHDRISFVQTLSGTGALAVAAKFLALFISRDIWIPDPSWANHKNIFQNNGFENIYRYSYYKDGQIDIDGWIEQLKTFAYNNQQENNKNPPCIILHACCHNPTGLDPTKEQWEKIIDTIYELKMVPIVDMAYQGLESGNLLKDAYLLRLCLNVNKYPNWSNGIFLCQSFAKNMGLYGERVGSLSVITPATANNGKFNPLQQKNSLQQNIDSQLKKIVRGMYSSPPGYGSRVVNVVLSDFKLKQQWFKDVDFMVQRLHHVRQEMFDRLGWPDLVNFAQQHGMFYYTRFSPKQVEILRNNYFVYLTGDGRLSLSGVNDSNVDYLCESLEAVSKMDK LAPichia stipitis AAT1 (Ps) amino acid sequence:

(SEQ ID NO: 9) MYRTSLLKQTARPSVRVSTRQFSVLNNQVRKWSEIPLAPPDKILGISEAYNKDANTSKINLGVGAYRDNSGKPIIFPSVKEAEKILLASEVEKEYTGITGSKKFQNAVKGFVFNNSGKDVNGQQLIEQNRIVTAQTISGTGSLRVIGDFLNRFYTNKKLLVPKPTWANHVAVFKDAGLEPEFYAYYETSKNDLDFANLKKSLSSQPDGSIVLLHACCHNPTGMDLTPEQWEEVLAIVQEKNFYPLVDMAYQGFASGNPYKDIGLIRRLNELVVQNKLKSYALCQSFAKNMGLYGERTGSISIITESAEASQAIESQLKKLIRPIYSSPPIHGSKIVEIIFDEQHNLLNSWLQDLDKVVGRLNTVRSKLYENLDKSSYNWDHLLKQRGMFVYTGLSAEQVIKLRNDYSVYATEDGRFSISGINDNNVEYLANAINEVVKQSaccharomyces cerevisiae AAT2 amino acid sequence:

(SEQ ID NO: 10) MSATLFNNIELLPPDALFGIKQRYGQDQRATKVDLGIGAYRDDNGKPWVLPSVKAAEKLIHNDSSYNHEYLGITGLPSLTSNAAKIIFGTQSDAFQEDRVISVQSLSGTGALHISAKFFSKFFPDKLVYLSKPTWANHMAIFENQGLKTATYPYWANETKSLDLNGFLNAIQKAPEGSIFVLHSCAHNPTGLDPTSEQWVQIVDAIASKNHIALFDTAYQGFATGDLDKDAYAVRLGVEKLSTVSPVFVCQSFAKNAGMYGERVGCFHLALTKQAQNKTIKPAVTSQLAKIIRSEVSNPPAYGAKIVAKLLETPELTEQWHKDMVTMSSRITKMRHALRDHLVKLGTPGNWDHIVNQCGMFSFTGLTPQMVKRLEETHAVYLVASGRASIAGLNQGNVEY VAKAIDEVVRFYTIEAKL

In particular embodiments of the invention, the polypeptide catalyzingthe conversion of L-aspartate to L-aspartyl-4-phospate is derived from ayeast. An example of a suitable source for this enzyme is the genusPichia, a preferred source is Picihia stipitis. Another example of asuitable source is the appropriate gene derived from the genusSaccharomyces, a preferred source is S. cerevisiae. The inventionfurther contemplates the use of an aspartate kinase that has beenmodified to become resistant to feedback inhibition.

A specific example of a suitable sequence is:

Pichia stipitis HOM3^(FBR) (Ps) amino acid sequence:

(SEQ ID NO: 11) MSVSPPLSAKSYNSIVDLRFTASKPQGWVVQKFGGTSVGKFPENIVDDIVLVFSKTNRVAVVCSARSSQTKSEGTTSRLLKAADIAAESGDFQYMLDVIEDDHVKNAEARVKNKTIQQKLVADTKREIAHAAELLRACQVIGEISARSLDSVMSIGEKLSCLFMAALMNDHGLKAVYIDLSDVIPLDYDFTNGFDDNFYKFLSQQLSSRALALSEDTVPVLTGYFGTVPGGLLNGVGRGYTDLCAALVAVGVQADELQVWKEVDGIFTADPRKVPTARLLDSVTPEEAAELTYYGSEVIHPFTMEQVIKAKIPIRIKNVVNPKGSGTIIFPDNVGRRGEETPPHPPEAYETLSSSFVLSHKKRSATAITAKQDIVVINIHSNKKTLSHGFLAHIFTTLDNFKLVVDLISTSEVHVSMALQILQDQELQLKNALKDLRRMGTVDITRNMTIISLVGKQMVNFIDIAGNMFKVLADNRINIEMISQGANEINISAVINEKDTIRALQSIHAKLLEGTFGFDDHVESAVDLRLESLKFQ

Another exemplary sequence is:

Saccharomyces cerevisiae HOM3^(FBR) amino acid sequence:

(SEQ ID NO: 12) MSVSPPLSAKSYNSIVDLRFTASKPQGWVVQKFGGTSVGKFPENIVDDIVLVFSKTNRVAVVCSARSSQTKSEGTTSRLLKAADIAAESGDFQYMLDVIEDDHVKNAEARVKNKTIQQKLVADTKREIAHAAELLRACQVIGEISARSLDSVMSIGEKLSCLFMAALMNDHGLKAVYIDLSDVIPLDYDFTNGFDDNFYKFLSQQLSSRALALSEDTVPVLTGYFGTVPGGLLNGVGRGYTDLCAALVAVGVQADELQVWKEVDGIFTADPRKVPTARLLDSVTPEEAAELTYYGSEVIHPFTMEQVIKAKIPIRIKNVVNPKGSGTIIFPDNVGRRGEETPPHPPEAYETLSSSFVLSHKKRSATAITAKQDIVVINIHSNKKTLSHGFLAHIFTTLDNFKLVVDLISTSEVHVSMALQILQDQELQLKNALKDLRRMGTVDITRNMTIISLVGKQMVNFIDIAGNMFKVLADNRINIEMISQGANEINISAVINEKDTIRALQSIHAKLLEGTFGFDDHVESAVDLRLESLKFQ

In particular embodiments of the invention, the polypeptide catalyzingthe conversion of L-aspartyl-4-phospate to 2-amino-4-oxo-butanoic acid(L-aspartate semialdehyde) is derived from a yeast. An example of asuitable source for this enzyme is the genus Pichia, a preferred sourceis Picihia stipitis.

A specific example of a suitable sequence is:

Pichia stipitis HOM2 (Ps) amino acid sequence:

(SEQ ID NO: 13) MVKKAGVLGATGSVGQRFILLLAEHPDFELHVLGASPRSAGKQYKDAVQWKQTDLLPENAQKIIVSECKAEAFKDCDIVFSGLDADYAGPIEKEFVEAGLVVVSNAKNYRREPGVPLIVPIVNSEHLSVIERKLAVAKAEGKSKPGYIICISNCSTAGLVAPLKPLIDAFGPIDALTATTLQAISGAGFSPGVPGMDVLDNIIPYIGGEEEKLEWESKKILGNLTKDGTDFAPLSNDEMKVSAQCNRVAVIDGHTECISFRFAKHPAPSVAQVKKVLSEYVCEATKLGCHSAPKQTIHVLEQQDRPQPRLDRNRDNGYGVSVGRIREDAVLDFKMVVLSHNTIIGAAGAG VLIAEILKAKDMI

Another exemplary sequence is:

Saccharomyces cerevisiae HOM2 (Sc) amino acid sequence:

(SEQ ID NO: 14) MAGKKIAGVLGATGSVGQRFILLLANHPHFELKVLGASSRSAGKKYVDAVNWKQTDLLPESATDIIVSECKSEFFKECDIVFSGLDADYAGAIEKEFMEAGIAIVSNAKNYRREQDVPLIVPVVNPEHLDIVAQKLDTAKAQGKPRPGFIICISNCSTAGLVAPLKPLIEKFGPIDALTTTTLQAISGAGFSPGVPGIDILDNIIPYIGGEEDKMEWETKKILAPLAEDKTHVKLLTPEEIKVSAQCNRVAVSDGHTECISLRFKNRPAPSVEQVKTCLKEYVCDAYKLGCHSAPKQTIHVLEQPDRPQPRLDRNRDSGYGVSVGRIREDPLLDFKMVVLSHNTIIGAAG SGVLIAEILLARNLI

In particular embodiments of the invention, the polypeptide catalyzingthe conversion of 2-amino-4-oxo-butanoic acid (L-aspartate semialdehyde)to homoserine is derived from a yeast. An example of a suitable sourcefor this enzyme is the genus Pichia, a preferred source is Picihiastipitis.

A specific example of a suitable sequence is:

Pichia stipitis HOM6 (Ps) amino acid sequence:

(SEQ ID NO: 15) MSKSVNVAIIGSGVVGSAFISQLNGLKTAIKYNVVYLAKTSSEALYSSDYQSVDLSSYKTSATKPTLGLDELLKFLQGAKKATILVDNTSNASIADYYPTFIKAGISIATPNKKAFSSDLKTWNEIFANSAVPGAGLVAHEATVGAGLPIIGPLRDLITTGDKVDKIEGIFSGTLSYIFNEFSTTEKSDVKFSDVVKVAKKLGYTEPDPRDDLNGLDVARKVTILARISGFEVESPTSFPVESLIPKELEGIESAAEFLEKLPNYDADIQKIKDEAFAENKTLRFVGQVDFKANKVSVGIGKYPFDHPFSALKGSDNVISIKTERYPNPLIVQGAGAGSEVTAHGVLADT IKIAERIAN

Another exemplary sequence is:

Saccharomyces cerevisiae HOM6 amino acid sequence:

(SEQ ID NO: 16) MSTKVVNVAVIGAGVVGSAFLDQLLAMKSTITYNLVLLAEAERSLISKDFSPLNVGSDWKAALAASTTKTLPLDDLIAHLKTSPKPVILVDNTSSAYIAGFYTKFVENGISIATPNKKAFSSDLATWKALFSNKPTNGFVYHEATVGAGLPIISFLREIIQTGDEVEKIEGIFSGTLSYIFNEFSTSQANDVKFSDVVKVAKKLGYTEPDPRDDLNGLDVARKVTIVGRISGVEVESPTSFPVQSLIPKPLESVKSADEFLEKLSDYDKDLTQLKKEAATENKVLRFIGKVDVATKSVSVGIEKYDYSHPFASLKGSDNVISIKTKRYTNPVVIQGAGAGAAVTAAGVLG DVIKIAQRL

In particular embodiments of the invention, the polypeptide catalyzingthe conversion of homoserine to O-phospho-L-homoserine is derived from ayeast. An example of a suitable source for this enzyme is the genusPichia, a preferred source is Picihia stipitis.

A specific example of a suitable sequence is:

Pichia stipitis THR1 (Ps) amino acid sequence:

(SEQ ID NO: 17) MTIRSFEVKVPASSANIGPGFDVLGVGLQLYLQIKVTIDSSKDTSHDPYHVKLSYEGDLAEKVPLTSDKNLITQTALYILRVNGMDSFPQGTHIHVINPVPLGRGLGSSASAIVGGIVLGNEIGEFKFSKTRLMDYCLMIERHPDNIAAAMLGGFVGSYLHDLSPEDMAAKNVPLDYILPKPDTPKEKIVSSQPPTNIGEYLQYNWCHKIKCVAIVPNFEVSTDSSRAVLPEKYDRQDIVFNLQRLAILTNALTQETPNNKLIYESMKDKIHQPYRSGLIPGLQKVLASVTPDTHPGLCGICLSGAGPTILCLATGGYDAIAETVIGIFNKAGVECSWKLLELAYDGATV EIK

Other exemplary sequences are:

Saccharomyces cerevisiae THR1 amino acid sequence:

(SEQ ID NO: 18) MVRAFKIKVPASSANIGPGYDVLGVGLSLFLELDVTIDSSQAQETNDDPNNCKLSYTKESEGYSTVPLRSDANLITRTALYVLRCNNIRNFPSGTKVHVSNPIPLGRGLGSSGAAVVAGVILGNEVAQLGFSKQRMLDYCLMIERHPDNITAAMMGGFCGSFLRDLTPQEVERREIPLAEVLPEPSGGEDTGLVPPLPPTDIGRHVKYQWNPAIKCIAIIPQFELSTADSRGVLPKAYPTQDLVFNLQRLAVLTTALTMDPPNADLIYPAMQDRVHQPYRKTLIPGLTEILSCVTPSTYPGLLGICLSGAGPTILALATENFEEISQEIINRFAKNGIKCSWKLLEPAYD GASVEQQCorynebacterium glutamicum KhsE (Cg) amino acid sequence:

(SEQ ID NO: 19) MAIELPVGKKVTVTVPASSANLGPGFDTLGLALSLYDTVEVEVTDHGLEVEVFGEGQGELPLDGSHLVVKAIRAGLKAADVQVPGLRVVCHNNIPQSRGLGSSAAAAVAGVAAANGLAGFPLDDARVVQLSSAFEGHPDNAAASVLGNAVVSWTEIPVDGRTEPQFKAVTINVDSRIKATALVPDFHASTEAVRRVLPSDVTHLDARFNVSRCAVMTVALQHHPELLWEGTRDRLHQPYRADVLPVTAEWVNRLRNRGYAAYLSGAGPTIMVLHTEPVDEAVLNDAREAGLRVLSLDVAD AVSVKVDA

In particular embodiments of the invention, the polypeptide catalyzingthe conversion of O-phospho-L-homoserine to L-threonine is derived froma yeast. An example of a suitable source for this enzyme is the genusPichia, a preferred source is Picihia stipitis.

A specific example of a suitable sequence is:

Pichia stipitis THR4 (Ps) amino acid sequence:

(SEQ ID NO: 20) MSQKYRSSRSAEPQALSFEDVVMTGLANDGGLFLPSQVPQLPASFLQDWADLSFQELAFNVLRLYINAAEIPDQDLRDLISKSYSTFRSEEVTPLKKIDDKLYLLELFHGPTYAFKDVALQFVGNLFEYFLTRRNAKKVEGEARDVITVVGATSGDTGSAAIYGLRGKKDVSVFILYPTGRISPIQEEQMTTVEDANVHTLSVNGTFDDCQDIVKSIFGDREFNDKYHVGAVNSINWARILAQQTYYFYSYFQLQKKLNDTSAKVRFVVPSGNFGDILAGYYAYKMGLPVDKLIIATNENDILDRFMKTGRYEKKAEKDASAAVKATFSPAMDILISSNFERLLWYLIRDSVANGSDEVAGKTLNSWMQQLKETGSVVADPEVLAGARSIFDSERVDDAETVATIKEVYSAHPESYVLDPHSSVGVTTSYRFIKKDDKKDNIKYISLSTAHPAKFSEVVNKALDSIAGYSFEKDVLPAELKALSTKRKRINLIDEASIEK VKDAIKKELNF

Another exemplary sequence is:

Saccharomyces cerevisiae THR4 amino acid sequence:

(SEQ ID NO: 21) MPNASQVYRSTRSSSPKTISFEEAIIQGLATDGGLFIPPTIPQVDQATLFNDWSKLSFQDLAFAIMRLYIAQEEIPDADLKDLIKRSYSTFRSDEVTPLVQNVTGDKENLHILELFHGPTYAFKDVALQFVGNLFEYFLQRTNANLPEGEKKQITVVGATSGDTGSAAIYGLRGKKDVSVFILYPTGRISPIQEEQMTTVPDENVQTLSVTGTFDNCQDIVKAIFGDKEFNSKHNVGAVNSINWARILAQMTYYFYSFFQATNGKDSKKVKFVVPSGNFGDILAGYFAKKMGLPIEKLAIATNENDILDRFLKSGLYERSDKVAATLSPAMDILISSNFERLLWYLAREYLANGDDLKAGEIVNNW*FQELKTNGKFQVDKSIIEGASKDFTSERVSNEETSETIKKIYESSVNPKHYILDPHTAVGVCATERLIAKDNDKSIQYISLSTAHPAKFADAVNNALSGFSNYSFEKDVLPEELKKLSTLKKKLKFIERADVE LVKNAIEEELAKMKL

A second aspect of the invention provides a recombinant microorganismcomprising at least one DNA molecule, wherein said at least one DNAmolecule encodes at least two polypeptides that catalyze a substrate toproduct conversion selected from the group consisting of steps i)through l), wherein said recombinant microorganism produces2-methylbutanol.

In particular embodiments of the invention, the polypeptide catalyzingthe conversion of L-threonine to 2-oxobutanate is derived from a yeast.An example of a suitable source for this enzyme is the genus Pichia, apreferred source is Picihia stipitis. The invention further contemplatesthe use of a threonine deaminase or threonine dehydratase that has beenmodified to become resistant to feedback inhibition.

A specific example of a suitable sequence is:

Pichia stipitis ILV1 (Ps)^(FBR) amino acid sequence:

(SEQ ID NO: 22) MFFSRSGEVEKFPNLLDADFNEDGDPDYIKLILTSRVYDVVERAGTPLTHAINLSHKCNSNIYLKREDLLPVFSFKLRGAYNMISHLHSNSKMPLSGVIACSAGNHAQGVAYSANRLKIPSTIVMPTATPSIKYTNVSRLGSQVVLYGDDFDSAKQECARLSSLNNLTDVPPFDHPYVIAGQGTIALEITRQLRLDKLNALFVPVGGGGLIAGVAVYLKKIAPHVKIIGVETNDADALYQSLKAKKLVVLDQVGMFADGTAVKVLGKETWRLCENLVDEVVKVSTDELCAAIKDIFEDTRLITEPSGALSVAGLKKYIEQNPDIDHRNKFYVPILSGANMNFDRLRFVSERAVLGEGKEVSLVVTIPEKPGEFAKLQSIINPRAITEFSYRCNGADANIFVSFNVIDKKKELTPIIEDMNNNEHGYEVVDISDNELAKTHGCYLVGGKSSEEVANERLYSFEFPEKPGALFNFLQALKADWNITLFHYHNHGHDIGKVLCGFTLPEGTDDADFQSFLNELGYKFNVENDNVVYKKFLRS

Another exemplary sequence that contemplates the use of a threoninedeaminase that has been modified to optimize cytoplasmic expression is:

Pichia stipitis ILV1 (Ps) Δ15 amino acid sequence:

(SEQ ID NO: 23) MFPNLLDADFNEDGDPDYIKLILTSRVYDVVERAGTPLTHAINLSHKCNSNIYLKREDLLPVFSFKLRGAYNMISHLHSNSKMPLSGVIACSAGNHAQGVAYSANRLKIPSTIVMPTATPSIKYTNVSRLGSQVVLYGDDFDSAKQECARLSSLNNLTDVPPFDHPYVIAGQGTIALEITRQLRLDKLNALFVPVGGGGLIAGVAVYLKKIAPHVKIIGVETNDADALYQSLKAKKLVVLDQVGMFADGTAVKVLGKETWRLCENLVDEVVKVSTDELCAAIKDIFEDTRLITEPSGALSVAGLKKYIEQNPDIDHRNKFYVPILSGANMNFDRLRFVSERAVLGEGKEVSLVVTIPEKPGEFAKLQSIINPRAITEFSYRCNGADANIFVSFNVIDKKKELTPIIEDMNNNEHGYEVVDISDNELAKTHGCYLVGGKSSEEVANERLYSFEFPEKPGALFNFLQALKADWNITLFHYHNHGHDIGKVLCGFTLPEGTDDADFQSFLNELGYKFNVENDNVVYKKFLRS

Other exemplary sequences are:

Saccharomyces cerevisiae ILV1 amino acid sequence:

(SEQ ID NO: 24) MSATLLKQPLCTVVRQGKQSKVSGLNLLRLKAHLHRQHLSPSLIKLHSELKLDELQTDNTPDYVRLVLRSSVYDVINESPISQGVGLSSRLNTNVILKREDLLPVFSFKLRGAYNMIAKLDDSQRNQGVIACSAGNHAQGVAFAAKHLKIPATIVMPVCTPSIKYQNVSRLGSQVVLYGNDFDEAKAECAKLAEERGLTNIPPFDHPYVIAGQGTVAMEILRQVRTANKIGAVFVPVGGGGLIAGIGAYLKRVAPHIKIIGVETYDAATLHNSLQRNQRTPLPVVGTFADGTSVRMIGEETFRVAQQVVDEVVLVNTDEICAAVKDIFEDTRSIVEPSGALSVAGMKKYISTVHPEIDHTKNTYVPILSGANMNFDRLRFVSERAVLGEGKEVFMLVTLPDVPGAFKKMQKIIHPRSVTEFSYRYNEHRHESSSEVPKAYIYTSFSVVDREKEIKQVMQQLNALGFEAVDISDNELAKSHGRYLVGGASKVPNERIISFEFPERPGALTRFLGGLSDSWNLTLFHYRNHGADIGKVLAGISVPPRENLTFQKFLEDLGYTYHDETDNTVYQKFLKY

Saccharomyces cerevisiae ILV1^(FBR) amino acid sequence:

(SEQ ID NO: 25) MSATLLKQPLCTVVRQGKQSKVSGLNLLRLKAHLHRQHLSPSLIKLHSELKLDELQTDNTPDYVRLVLRSSVYDVINESPISQGVGLSSRLNTNVILKREDLLPVFSFKLRGAYNMIAKLDDSQRNQGVIACSAGNHAQGVAFAAKHLKIPATIVMPVCTPSIKYQNVSRLGSQVVLYGNDFDEAKAECAKLAEERGLTNIPPFDHPYVIAGQGTVAMEILRQVRTANKIGAVFVPVGGGGLIAGIGAYLKRVAPHIKIIGVETYDAATLHNSLQRNQRTPLPVVGTFADGTSVRMIGEETFRVAQQVVDEVVLVNTDEICAAVKDIFEDTRSIVEPSGALSVAGMKKYISTVHPEIDHTKNTYVPILSGANMNFDRLRFVSERAVLGEGKEVFMLVTLPDVPGAFKKMQKIIHPRSVTEFSYRYNEHRHESSSEVPKAYIYTSFSVVDREKEIKQVMQQLNALGFEAVDISDNELAKSHGCYLVGGASKVPNERIISFEFPERPGALTRFLGGLSDSWNLTLFHYHNHGADIGKVLAGISVPPRENLTFQKFLEDLGYTYHDETDNTVYQKFLKY

Pichia stipitis ILV1 (Ps) amino acid sequence:

(SEQ ID NO: 26) MFFSRSGEVEKFPNLLDADFNEDGDPDYIKLILTSRVYDVVERAGTPLTHAINLSHKCNSNIYLKREDLLPVFSFKLRGAYNMISHLHSNSKMPLSGVIACSAGNHAQGVAYSANRLKIPSTIVMPTATPSIKYTNVSRLGSQVVLYGDDFDSAKQECARLSSLNNLTDVPPFDHPYVIAGQGTIALEITRQLRLDKLNALFVPVGGGGLIAGVAVYLKKIAPHVKIIGVETNDADALYQSLKAKKSVVLDQVGMFADGTAVKVLGKETWRLCENLVDEVVKVSTDELCAAIKDIFEDTRSITEPSGALSVAGLKKYIEQNPDIDHRNKFYVPILSGANMNFDRLRFVSERAVLGEGKEVSLVVTIPEKPGEFAKLQSIINPRAITEFSYRCNGADANIFVSFNVIDKKKELTPIIEDMNNNEHGYEVVDISDNELAKTHGRYLVGGKSSEEVANERLYSFEFPEKPGALFNFLQALKADWNITLFHYRNHGHDIGKVLCGFTLPEGTDDADFQSFLNELGYKFNVENDNVVYKKFLRS

Saccharomyces cerevisiae CHA1 amino acid sequence:

(SEQ ID NO: 27) MSIVYNKTPLLRQFFPGKASAQFFLKYECLQPSGSFKSRGIGNLIMKSAIRIQKDGKRSPQVFASSGGNAGFAAATACQRLSLPCTVVVPTATKKRMVDKIRNTGAQVIVSGAYWKEADTFLKTNVMNKIDSQVIEPIYVHPFDNPDIWEGHSSMIDEIVQDLKSQHISVNKVKGIVCSVGGGGLYNGIIQGLERYGLADRIPIVGVETNGCHVFNTSLKIGQPVQFKKITSIATSLGTAVISNQTFEYARKYNTRSVVIEDKDVIETCLKYTHQFNMVIEPACGAALHLGYNTKILENALGSKLAADDIVIIIACGGSSNTIKDLEEALDSMRKKDTPVIEVADNFIFP EKNIVNLKSA

Corynebacterium glutamicum IlvA (Cg) amino acid sequence:

(SEQ ID NO: 28) MSETYVSEKSPGVMASGAELIRAADIQTAQARISSVIAPTPLQYCPRLSEETGAEIYLKREDLQDVRSYKIRGALNSGAQLTQEQRDAGIVAASAGNHAQGVAYVCKSLGVQGRIYVPVQTPKQKRDRIMVHGGEFVSLVVTGNNFDEASAAAHEDAERTGATLIEPFDARNTVIGQGTVAAEILSQLTSMGKSADHVMVPVGGGGLLAGVVSYMADMAPRTAIVGIEPAGAASMQAALHNGGPITLETVDPFVDGAAVKRVGDLNYTIVEKNQGRVHMMSATEGAVCTEMLDLYQNEGIIAEPAGALSIAGLKEMSFAPGSVVVCIISGGNNDVLRYAEIAERSLVHRGLKHYFLVNFPQKPGQLRHFLEDILGPDDDITLFEYLKRNNRETGTALVGIHLSEASGLDSLLERMEESAIDSRRLEPGTPEYEYLT

Escherichia coli ilvA (Ec) amino acid sequence:

(SEQ ID NO: 29) MADSQPLSGAPEGAEYLRAVLRAPVYEAAQVTPLQKMEKLSSRLDNVILVKREDRQPVHSFKLRGAYAMMAGLTEEQKAHGVITASAGNHAQGVAFSSARLGVKALIVMPTATADIKVDAVRGFGGEVLLHGANFDEAKAKAIELSQQQGFTWVPPFDHPMVIAGQGTLALELLQQDAHLDRVFVPVGGGGLAAGVAVLIKQLMPQIKVIAVEAEDSACLKAALDAGHPVDLPRVGLFAEGVAVKRIGDETFRLCQEYLDDIITVDSDAICAAMKDLFEDVRAVAEPSGALALAGMKKYIAQHNIRGERLAHILSGANVNFHGLRYVSERCELGEQREALLAVTIPEEKGSFLKFCQLLGGRSVTEFNYRFADAKNACIFVGVRLSRGLEERKEILQMLNDGGYSVVDLSDDEMAKLHVRYMVGGRPSHPLQERLYSFEFPESPGALLRFLNTLGTHWNISLFHYRSHGTDYGRVLAAFELGDHEPDFETRLNELGYDCH DETINPAFRFFLAG

In particular embodiments of the invention, the polypeptide catalyzingthe conversion of 2-oxobutanate to 2-aceto-hydroxy-butyrate is derivedfrom a yeast. An example of a suitable source for this enzyme is thegenus Pichia, a preferred source is Picihia stipitis.

A specific example of a suitable sequence is:

Pichia stipitis ILV2 (Ps) amino acid sequence:

(SEQ ID NO: 30) MARAALSRSSGSRYAIRALSNTKLHNATMSATSRPTPSPAFNAADIRQPQSYPTQRKKNDFVMDDSFIGLTGGEIFHEMMLRHNVDTVFGYAGGAILPVFDAIYNSDKFKFVLPRHEQGAGHMAEGYARATGKPGVVLVTSGPGATNVITPLADALMDGVPLVVFTGQVPTTAIGTDAFQEADVVGISRSCTKWNVMVKNVAELPRRINEAFEIATSGRPGPVLVDLPKDVTAAILREAIPINSTLPSNALQQITKEAQNEFTMGAIARSANLLNVAKKPIIYAGAGVLNHEDGPKLLKELSDKANIPVTTTLQGLGAFDQRDPKSLDMLGMHGHAAANTAMQDADCIIALGARFDDRVTGNINKFAPEAKLAASEGRGGIIHFEISPKNINKVVEATEAVEGDLTANLRSFIPLVKPVAERPQWLGKINEWKEKYPYAYQLETPGSLIKPQTLIKEISEQSSTYNKEVIVTTGVGQHQMWAAQHFTWTKPRTMITSGGLGTMGYGLPAAIGAQIGKPDAIVIDIDGDASFNMTLTELSSAVQAGAPVKVCVLNNEEQGMVTQWQSLFYEHRYSHTHQSNPDFMKLADAMGVQGIRISTQEELKSGVKAFLDAKGPVLLEVIVEKKVPVLPMVPAGSALDDFILWDAETE KQQKELRNERTGGKH

Another exemplary sequence that contemplates the use of An acetolactatesynthase that has been modified to optimize cytoplasmic expression is:

Pichia stipitis ILV2 (Ps)A26 amino acid sequence:

(SEQ ID NO: 31) MATMSATSRPTPLPAFNAADIRQPQSYPTQRKKNDFVMDDSFIGLTGGEIFHEMMLRHNVDTVFGYAGGAILPVFDAIYNSDKFKFVLPRHEQGAGHMAEGYARATGKPGVVLVTSGPGATNVITPLADALMDGVPLVVFTGQVPTTAIGTDAFQEADVVGISRSCTKWNVMVKNVAELPRRINEAFEIATSGRPGPVLVDLPKDVTAAILREAIPINSTLPSNALQQITKEAQNEFTMGAIARSANLLNVAKKPIIYAGAGVLNHEDGPKLLKELSDKANIPVTTTLQGLGAFDQRDPKSLDMLGMHGHAAANTAMQDADCIIALGARFDDRVTGNINKFAPEAKLAASEGRGGIIHFEISPKNINKVVEATEAVEGDLTANLRSFIPLVKPVAERPQWLGKINEWKEKYPYAYQLETPGSLIKPQTLIKEISEQSSTYNKEVIVTTGVGQHQMWAAQHFTWTKPRTMITSGGLGTMGYGLPAAIGAQIGKPDAIVIDIDGDASFNMTLTELSSAVQAGAPVKVCVLNNEEQGMVTQWQSLFYEHRYSHTHQSNPDFMKLADAMGVQGIRISTQEELKSGVKAFLDAKGPVLLEVIVEKKVPVLPMVPAGSALDDFILWDAETEKQQKELRNERTGGKH

Other exemplary sequences are:

Saccharomyces cerevisiae ILV2 amino acid sequence:

(SEQ ID NO: 32) MIRQSTLKNFAIKRCFQHIAYRNTPAMRSVALAQRFYSSSSRYYSASPLPASKRPEPAPSFNVDPLEQPAEPSKLAKKLRAEPDMDTSFVGLTGGQIFNEMMSRQNVDTVFGYPGGAILPVYDAIHNSDKFNFVLPKHEQGAGHMAEGYARASGKPGVVLVTSGPGATNVVTPMADAFADGIPMVVFTGQVPTSAIGTDAFQEADVVGISRSCTKWNVMVKSVEELPLRINEAFEIATSGRPGPVLVDLPKDVTAAILRNPIPTKTTLPSNALNQLTSRAQDEFVMQSINKAADLINLAKKPVLYVGAGILNHADGPRLLKELSDRAQIPVTTTLQGLGSFDQEDPKSLDMLGMHGCATANLAVQNADLIIAVGARFDDRVTGNISKFAPEARRAAAEGRGGIIHFEVSPKNINKVVQTQIAVEGDATTNLGKMMSKIFPVKERSEWFAQINKWKKEYPYAYMEETPGSKIKPQTVIKKLSKVANDTGRHVIVTTGVGQHQMWAAQHWTWRNPHTFITSGGLGTMGYGLPAAIGAQVAKPESLVIDIDGDASFNMTLTELSSAVQAGTPVKILILNNEEQGMVTQWQSLFYEHRYSHTHQLNPDFIKLAEAMGLKGLRVKKQEELDAKLKEFVSTKGPVLLEVEVDKKVPVLPMVAGGSGLDEFINFDPEVERQQTELRHKRTGGKH

Saccharomyces cerevisiae ILV6 amino acid sequence:

(SEQ ID NO: 33) MLRSLLQSGHRRVVASSCATMVRCSSSSTSALAYKQMHRHATRPPLPTLDTPSWNANSAVSSIIYETPAPSRQPRKQHVLNCLVQNEPGVLSRVSGTLAARGFNIDSLVVCNTEVKDLSRMTIVLQGQDGVVEQARRQIEDLVPVYAVLDYTNSEIIKRELVMARISLLGTEYFEDLLLHHHTSTNAGAADSQELVAEIREKQFHPANLPASEVLRLKHEHLNDITNLTNNFGGRVVDISETSCIVELSAKPTRISAFLKLVEPFGVLECARSGMMALPRTPLKTSTEEAADED EKISEIVDISQLPPG

Pichia stipitis LV6 (Ps) amino acid sequence:

(SEQ ID NO: 34) MFAKQTLRRSASSAYKQGVRNKQTSSSTSALAYKTLHRNQKRPPLPTLETPNWSADAAVSSILYETPMPSKAPRKQHVLNCLVQNEPGVLSSVSGTLAARGFNIDSLVVCNTEVKDLSRMTIVLAGQDAVVEQARRQIEDLVPVYAVLDYTNAEIIKRELLLARVSLLGPEYFQELIATHKLHISDGSAVPDLSATDSAYHPNNLAPSEALRQKHIHLDHINTITEKFGGKIVDLSDRNVIVELSAKPSRITSFLHLLQPFGILELARSGMMALPRTPLDAAVEEDEPVEAADV VDASQLPPG

Corynebacterium glutamicum IlvB (Cg) amino acid sequence:

(SEQ ID NO: 35) MNVAASQQPTPATVASRGRSAAPERMTGAKAIVRSLEELNADIVFGIPGGAVLPVYDPLYSSTKVRHVLVRHEQGAGHAATGYAQVTGRVGVCIATSGPGATNLVTPIADANLDSVPMVAITGQVGSGLLGTDAFQEADIRGITMPVTKHNFMVTNPNDIPQALAEAFHLAITGRPGPVLVDIPKDVQNAELDFVWPPKIDLPGYRPVSTPHARQIEQAVKLIGEAKKPVLYVGGGVIKADAHEELRAFAEYTGIPVVTTLMALGTFPESHELHMGMPGMHGTVSAVGALQRSDLLIAIGSRFDDRVTGDVDTFAPDAKIIHADIDPAEIGKIKQVEVPIVGDAREVLARLLETTKASKAETEDISEWVDYLKGLKARFPRGYDEQPGDLLAPQFVIETLSKEVGPDAIYCAGVGQHQMWAAQFVDFEKPRTWLNSGGLGTMGYAVPAALGAKAGAPDKEVWAIDGDGCFQMTNQELTTAAVEGFPIKIALINNGNLGMVRQWQTLFYEGRYSNTKLRNQGEYMPDFVTLSEGLGCVAIRVTKAEEVLPAIQKAREINDRPVVIDFIVGEDAQVWPMVSAGSSNSDIQYALGLRPFFDGDESAAEDPADIHEAVSDIDAAVESTEA

Corynebacterium glutamicum ilvN (Cg) amino acid sequence:

(SEQ ID NO: 36) MANSDVTRHILSVLVQDVDGIISRVSGMFTRRAFNLVSLVSAKTETHGINRITVVVDADELNIEQITKQLNKLIPVLKVVRLDEETTIARAIMLVKVSADSTNRPQIVDAANIFRARVVDVAPDSVVIESTGTPGKLRALLDVMEPFGIRELIQSGQIALNRGPKTMAPAKI

Escherichia coli ilvB(Ec) amino acid sequence:

(SEQ ID NO: 37) MASSGTTSTRKRFTGAEFIVHFLEQQGIKIVTGIPGGSILPVYDALSQSTQIRHILARHEQGAGFIAQGMARTDGKPAVCMACSGPGATNLVTAIADARLDSIPLICITGQVPASMIGTDAFQEVDTYGISIPITKHNYLVRHIEELPQVMSDAFRIAQSGRPGPVWIDIPKDVQTAVFEIETQPAMAEKAAAPAFSEESIRDAAAMINAAKRPVLYLGGGVINAPARVRELAEKAQLPTTMTLMALGMLPKAHPLSLGMLGMHGVRSTNYILQEADLLIVLGARFDDRAIGKTEQFCPNAKIIHVDIDRAELGKIKQPHVAIQADVDDVLAQLIPLVEAQPRAEWHQLVADLQREFPCPIPKACDPLSHYGLINAVAACVDDNAIITTDVGQHQMWTAQAYPLNRPRQWLTSGGLGTMGFGLPAAIGAALANPDRKVLCFSGDGSLMMNIQEMATASENQLDVKIILMNNEALGLVHQQQSLFYEQGVFAATYPGKINFMQIAAGFGLETCDLNNEADPQASLQEIINRPGPALIHVRIDAEEKVYPMVPPGAANTEMVGE

Escherichia coli ilvN (Ec) amino acid sequence:

(SEQ ID NO: 38) MQNTTHDNVILELTVRNHPGVMTHVCGLFARRAFNVEGILCLPIQDSDKSHIWLLVNDDQRLEQMISQIDKLEDVVKVQRNQSDPTMFNKIAVFFQ

Escherichia coli ilvG (Ec) amino acid sequence:

(SEQ ID NO: 39) MNGAQWVVHALRAQGVNTVFGYPGGAIMPVYDALYDGGVEHLLCRHEQGAAMAAIGYARATGKTGVCIATSGPGATNLITGLADALLDSIPVVAITGQVSAPFIGTDAFQEVDILGLSLACTKHSFLVQSLEELPRIMAEAFDVASSGRPGPVLVDIPKDIQLASGDLEPWFTTVENEVTFPHAEVEQARQMLAKAQKPMLYVGGGVGMAQAVSALREFLAATKMPATCTLKGLGAVEADYPYYLGMLGMHGTKAANFAVQECDLLIAVGARFDDRVTGKLNTFAPHASVIHMDIDPAEMNKLRQAHVALQGDLNALLPALQQPLNINDWQQYCAQLRDEHTWRYDHPGDAIYAPLLLKQLSDRKPADCVVTTDVGQHQMWAAQHIAHTRPENFITSSGLGTMGFGLPAAVGAQVARPNDTVVCISGDGSFMMNVQELGTVKRKQLPLKIVLLDNQRLGMVRQWQQLFFQERYSETTLTDNPDFLMLASAFGIPGQHITRKDQVEAALDTMLNSDGPYLLHVSIDELENVWPLVPPGAS NSEMLEKLS

Escherichia coli ilvM (Ec) amino acid sequence:

(SEQ ID NO: 40) MMQHQVNVSARFNPETLERVLRVVRHRGFHVCSMNMAAASDAQNINIELTVASPRSVDLLFSQLNKLVDVAHVAICQSTTTSQQIRA

Escherichia coli ilvI (Ec) amino acid sequence:

(SEQ ID NO: 41) MEMLSGAEMVVRSLIDQGVKQVFGYPGGAVLDIYDALHTVGGIDHVLVRHEQAAVHMADGLARATGEVGVVLVTSGPGATNAITGIATAYMDSIPLVVLSGQVATSLIGYDAFQECDMVGISRPVVKHSFLVKQTEDIPQVLKKAFWLAASGRPGPVVVDLPKDILNPANKLPYVWPESVSMRSYNPTTTGHKGQIKRALQTLVAAKKPVVYVGGGAITAGCHQQLKETVEALNLPVVCSLMGLGAFPATHRQALGMLGMHGTYEANMTMHNADVIFAVGVRFDDRTTNNLAKYCPNATVLHIDIDPTSISKTVTADIPIVGDARQVLEQMLELLSQESAHQPLDEIRDWWQQIEQWRARQCLKYDTHSEKIKPQAVIETLWRLTKGDAYVTSDVGQHQMFAALYYPFDKPRRWINSGGLGTMGFGLPAALGVKMALPEETVVCVTGDGSIQMNIQELSTALQYELPVLVVNLNNRYLGMVKQWQDMIYSGRHSQSYMQSLPDFVRLAEAYGHVGIQISHPHELESKLSEALEQVRNNRLVFVDVTVDGSEHVYPMQIRGGGMDEMWLSKTERT

Escherichia coli ilvIH(Ec) amino acid sequence:

(SEQ ID NO: 42) MRRILSVLLENESGALSRVIGLFSQRGYNIESLTVAPTDDPTLSRMTIQTVGDEKVLEQIEKQLHKLVDVLRVSELGQGAHVEREIMLVKIQASGYGRDEVKRNTEIFRGQIIDVTPSLYTVQLAGTSGKLSAFLASIRDVAKIVEV ARSGVVGLSRGDKIMR

In particular embodiments of the invention, the polypeptide catalyzingthe conversion of 2-aceto-hydroxy-butyrate to2,3-dihydroxy-3-methylvalerate is derived from a yeast. An example of asuitable source for this enzyme is the genus Pichia, a preferred sourceis Picihia stipitis.

A specific example of a suitable sequence is:

Pichia stipitis ILV5 (Ps) amino acid sequence:

(SEQ ID NO: 43) MSFRRSSLRMAKMASAAASKQIASKRAMSALASAAKPVVSKQSMAPLAVRGIKTINFGGTEEVVHERADWPREKLLEYFKNDTLALIGYGSQGYGQGLNLRDNGLNVIIGVRKNGASWKAAIEDGWVPGENLFDVNEAISKGTYIMNLLSDAAQSETWASIKPQLTEGKTLYFSHGFSPVFKELTHVEPPTNIDVILAAPKGSGRTVRSLFKEGRGINSSYAVWNDVTGKAEEKAIALAVAIGSGYVYQTTFEREVNSDLYGERGCLMGGIHGMFLAQYEVLRENGHTPSEAFNETVEEATQSLYPLIGKYGMDYMYDACSTTARRGALDWYPRFKDALKPVFNDLYESVKNGSETQRSLDFNSQPDYREKLEEELQVIRNMEIWRVGKEVR KLRPENQ

Another exemplary sequence that contemplates the use of a ketol-acidreductoismorease that has been modified to optimize cytoplasmicexpression is:

Pichia stipitis ILV5 (Ps) A40 amino acid sequence:

(SEQ ID NO: 44) MKQSMAPLAVRGIKTINFGGTEEVVHERADWPREKLLEYFKNDTLALIGYGSQGYGQGLNLRDNGLNVIIGVRKNGASWKAAIEDGWVPGENLFDVNEAISKGTYIMNLLSDAAQSETWASIKPQLTEGKTLYFSHGFSPVFKELTHVEPPTNIDVILAAPKGSGRTVRSLFKEGRGINSSYAVWNDVTGKAEEKAIALAVAIGSGYVYQTTFEREVNSDLYGERGCLMGGIHGMFLAQYEVLRENGHTPSEAFNETVEEATQSLYPLIGKYGMDYMYDACSTTARRGALDWYPRFKDALKPVFNDLYESVKNGSETQRSLDFNSQPDYREKLEEELQVIRNM EIWRVGKEVRKLRPENQ

Other exemplary sequences are:

Saccharomyces cerevisiae ILV5 amino acid sequence:

(SEQ ID NO: 45) MLRTQAARLICNSRVITAKRTFALATRAAAYSRPAARFVKPMITTRGLKQINFGGTVETVYERADWPREKLLDYFKNDTFALIGYGSQGYGQGLNLRDNGLNVIIGVRKDGASWKAAIEDGWVPGKNLFTVEDAIKRGSYVMNLLSDAAQSETWPAIKPLLTKGKTLYFSHGFSPVFKDLTHVEPPKDLDVILVAPKGSGRTVRSLFKEGRGINSSYAVWNDVTGKAHEKAQALAVAIGSGYVYQTTFEREVNSDLYGERGCLMGGIHGMFLAQYDVLRENGHSPSEAFNETVEEATQSLYPLIGKYGMDYMYDACSTTARRGALDWYPIFKNALKPVFQDLYESTKNGTETKRSLEFNSQPDYREKLEKELDTIRNMEIWKVGKEVRKLRP ENQ

Corynebacterium glutamicum IlvC (cg) amino acid sequence:

(SEQ ID NO: 46) MAIELLYDADADLSLIQGRKVAIVGYGSQGHAHSQNLRDSGVEVVIGLREGSKSAEKAKEAGFEVKTTAEAAAWADVIMLLAPDTSQAEIFTNDIEPNLNAGDALLFGHGLNIHFDLIKPADDIIVGMVAPKGPGHLVRRQFVDGKGVPCLIAVDQDPTGTAQALTLSYAAAIGGARAGVIPTTFEAETVTDLFGEQAVLCGGTEELVKVGFEVLTEAGYEPEMAYFEVLHELKLIVDLMFEGGISNMNYSVSDTAEFGGYLSGPRVIDADTKSRMKDILTDIQDGTFTKRLIANVENGNTELEGLRASYNNHPIEETGAKLRDLMSWVKVDARAETA

Escherichia coli ilvC (Ec) amino acid sequence:

(SEQ ID NO: 47) MANYFNTLNLRQQLAQLGKCRFMGRDEFADGASYLQGKKVVIVGCGAQGLNQGLNMRDSGLDISYALRKEAIAEKRASWRKATENGFKVGTYEELIPQADLVINLTPDKQHSDVVRTVQPLMKDGAALGYSHGFNIVEVGEQIRKDITVVMVAPKCPGTEVREEYKRGFGVPTLIAVHPENDPKGEGMAIAKAWAAATGGHRAGVLESSFVAEVKSDLMGEQTILCGMLQAGSLLCFDKLVEEGTDPAYAEKLIQFGWETITEALKQGGITLMMDRLSNPAKLRAYALSEQLKEIMAPLFQKHMDDIISGEFSSGMMADWANDDKKLLTWREETGKTAFETAPQYEGKIGEQEYFDKGVLMIAMVKAGVELAFETMVDSGIIEESAYYESLHELPLIANTIARKRLYEMNVVISDTAEYGNYLFSYACVPLLKPFMAELQPGDLGKAIPEGAVDNGQLRDVNEAIRSHAIEQVGKKLRGYMTDMKRIAVA G

In particular embodiments of the invention, the polypeptide catalyzingthe conversion of 2,3-dihydroxy-3-methylvalerate to2-keto-3-methylvalerate is derived from a yeast. An example of asuitable source for this enzyme is the genus Pichia, a preferred sourceis Picihia stipitis.

A specific example of a suitable sequence is:

Pichia stipitis ILV3 (Ps)amino acid sequence:

(SEQ ID NO: 48) MSFLFKAAAARRVASKSPAAVARSFSVSATQCEKKLNKYSSIVTGDPSQGASQAMLYATGFDDADFNRAQIGVGSVWWSGNPCNMHLMELNNKCTESVNRAGLKGMQFNSIGISDGITNGTEGMRYSLQSREIIADSFESMMLGQLYDGNIAIPSCDKNMPGVLIAMARHNRPSIMVYGGTILPGQTTCGTNNPAIADKIDIISAFQSYGQYLTKSITNEERKDIVRHACPGPGACGGMYTANTMASAAECLGMSLPYSSSAPAVSKEKDAECANIGQAIKHLLEIDLKPRDILTKKSFENAIAYIIATGGSTNAVLHLIAIASSADIDLTVDDFQRISDSTPLLADFKPSGQFVMADLQKYGGTPAVMKFLMNEGFIDGDQYTVTGKTIKENLASVKDLPADQPIIRPVSNPLKTSGHLQILKGSLAPGSAVGKITGKEGTYFKGKARVFDDEGDFIVALEKGEIKKGEKTVCVIRYEGPKGGPGMPEMLKPSSALMGYGLGKDVALLTDGRFSGGSHGFLIGHIVPEAAEGGPIGLVYDGDEIVIDAENNKIDLLVDEAVLAERRKLWTAPEPRYTRGTLAKYARL VSDASAGCVTDLPIKN

Another exemplary sequence that contemplates the use of a dihydroxyaciddehydratase that has been modified to optimize cytoplasmic expressionis:

Pichia stipitis ILV3 (Ps)Δ34 amino acid sequence:

(SEQ ID NO: 49) MKKLNKYSSIVTGDPSQGASQAMLYATGFDDADFNRAQIGVGSVWWSGNPCNMHLMELNNKCTESVNRAGLKGMQFNSIGISDGITNGTEGMRYSLQSREIIADSFESMMLGQLYDGNIAIPSCDKNMPGVLIAMARHNRPSIMVYGGTILPGQTTCGTNNPAIADKIDIISAFQSYGQYLTKSITNEERKDIVRHACPGPGACGGMYTANTMASAAECLGMSLPYSSSAPAVSKEKDAECANIGQAIKHLLEIDLKPRDILTKKSFENAIAYIIATGGSTNAVLHLIAIASSADIDLTVDDFQRISDSTPLLADFKPSGQFVMADLQKYGGTPAVMKFLMNEGFIDGDQYTVTGKTIKENLASVKDLPADQPIIRPVSNPLKTSGHLQILKGSLAPGSAVGKITGKEGTYFKGKARVFDDEGDFIVALEKGEIKKGEKTVCVIRYEGPKGGPGMPEMLKPSSALMGYGLGKDVALLTDGRFSGGSHGFLIGHIVPEAAEGGPIGLVYDGDEIVIDAENNKIDLLVDEAVLAERRKLWTAPEPRYTRGTLAKYARLVSDASAGCVTDLPIKN

Other exemplary sequences are:

Saccharomyces cerevisiae ILV3 amino acid sequence:

(SEQ ID NO: 50) MGLLTKVATSRQFSTTRCVAKKLNKYSYIITEPKGQGASQAMLYATGFKKEDFKKPQVGVGSCWWSGNPCNMHLLDLNNRCSQSIEKAGLKAMQFNTIGVSDGISMGTKGMRYSLQSREIIADSFETIMMAQHYDANIAIPSCDKNMPGVMMAMGRHNRPSIMVYGGTILPGHPTCGSSKISKNIDIVSAFQSYGEYISKQFTEEEREDVVEHACPGPGSCGGMYTANTMASAAEVLGLTIPNSSSFPAVSKEKLAECDNIGEYIKKTMELGILPRDILTKEAFENAITYVVATGGSTNAVLHLVAVAHSAGVKLSPDDFQRISDTTPLIGDFKPSGKYVMADLINVGGTQSVIKYLYENNMLHGNTMTVTGDTLAERAKKAPSLPEGQEIIKPLSHPIKANGHLQILYGSLAPGGAVGKITGKEGTYFKGRARVFEEEGAFIEALERGEIKKGEKTVVVIRYEGPRGAPGMPEMLKPSSALMGYGLGKDVALLTDGRFSGGSHGFLIGHIVPEAAEGGPIGLVRDGDEIIIDADNNKIDLLVSDKEMAQRKQSWVAPPPRYTRGTLSKYAKLVSNASNGCVLDA

Corynebacterium glutamicum IlvD (Cg) amino acid sequence:

(SEQ ID NO: 51) MIPLRSKVTTVGRNAAGARALWRATGTKENEFGKPIVAIVNSYTQFVPGHVHLKNVGDIVADAVRKAGGVPKEFNTIAVDDGIAMGHGGMLYSLPSREIIADSVEYMVNAHTADAMVCISNCDKITPGMLNAAMRLNIPVVFVSGGPMEAGKAVVVDGVAHAPTDLITAISASASDAVDDAGLAAVEASACPTCGSCSGMFTANSMNCLTEALGLSLPGNGSTLATHAARRALFEKAGETVVELCRRYYGEEDESVLPRGIATKKAFENAMALDMAMGGSTNTILHILAAAQEGEVDFDLADIDELSKNVPCLSKVAPNSDYHMEDVHRAGGIPALLGELNRGGLLNKDVHSVHSNDLEGWLDDWDIRSGKTTEVATELFHAAPGGIRTTEAFSTENRWDELDTDAAKGCIRDVEHAYTADGGLVVLRGNISPDGAVIKSAGIEEELWNFTGPARVVESQEEAVSVILTKTIQAGEVLVVRYEGPSGGPGMQEMLHPTAFLKGSGLGKKCALITDGRFSGGSSGLSIGHVSPEAAHGGVIGLIENGDIVSIDVHNRKLEVQVSDEELQRRRDAMNASEKPWQPVNRNRVVTKALRAYAKMATSADKGAVRQVD

Escherichia coli ilvD (Ec) amino acid sequence:

(SEQ ID NO: 52) MPKYRSATTTHGRNMAGARALWRATGMTDADFGKPIIAVVNSFTQFVPGHVHLRDLGKLVAEQIEAAGGVAKEFNTIAVDDGIAMGHGGMLYSLPSRELIADSVEYMVNAHCADAMVCISNCDKITPGMLMASLRLNIPVIFVSGGPMEAGKTKLSDQIIKLDLVDAMIQGADPKVSDSQSDQVERSACPTCGSCSGMFTANSMNCLTEALGLSQPGNGSLLATHADRKQLFLNAGKRIVELTKRYYEQNDESALPRNIASKAAFENAMTLDIAMGGSTNTVLHLLAAAQEAEIDFTMSDIDKLSRKVPQLCKVAPSTQKYHMEDVHRAGGVIGILGELDRAGLLNRDVKNVLGLTLPQTLEQYDVMLTQDDAVKNMFRAGPAGIRTTQAFSQDCRWDTLDDDRANGCIRSLEHAYSKDGGLAVLYGNFAENGCIVKTAGVDDSILKFTGPAKVYESQDDAVEAILGGKVVAGDVVVIRYEGPKGGPGMQEMLYPTSFLKSMGLGKACALITDGRFSGGTSGLSIGHVSPEAASGGSIGLIEDGDLIAIDIPNRGIQLQVSDAELAARREAQDARGDKAWTPKNRERQVSFALRAYASLATSADKGAVRDKSKLGG

A third aspect of the invention provides a recombinant microorganismcomprising at least one DNA molecule, wherein said at least one DNAmolecule encodes (i) a polypeptide that catalyzes a2-keto-3-methyl-valerate to 2-methylbutanal conversion, and (ii) apolypeptide that catalyzes a 2-methylbutanal to 2-methylbutanolconversion, wherein said recombinant microorganism produces2-methylbutanol.

In particular embodiments of the invention, the polypeptide catalyzingthe conversion of 2-keto-3-methylvalerate to 2-methylbutanal is derivedfrom a yeast. An example of a suitable source for this enzyme is thegenus Pichia, a preferred source is Pichia stipitis.

A specific example of a suitable sequence is:

Pichia stipitis PDC3-6 (Ps) amino acid sequence:

(SEQ ID NO: 53) MTPVQETIRLPGTSSPTVPENVTLGEYLFLRISQANPKLRSIFGIPGDFNVDLLEHLYSPVVAGRDIKFIGLCNELNGAYTADGYSRAIGGLSTFISTFGVGELSAINGIAGSFAEFSPVLHIVGTTSLPQRDHAINGSDVRNHHHLIQNKNPLCQPNHDVYKKMIEPISVIQESLDSDLQRNMEKIDRVLVKILQESRPGYLFIPCDITNLIVPSYRLYETPLPLEIQLTTSGVEVLEDVVDAILFRLYKSKNPSLLSDCLTTRFNLQDKLNTLVAKLPSNFVKLFSTNMARNIDESLSNFVGLYFGIGSSSKEVSRQLERNTDFLINLGYFNAETTTAGYSNDFSNIEEYIEINPDYIKVNEHIINIKNPESGKRLFSMGQLLDALLFKLDLNKIENINNNNISYKFFPPTLYEQDNNTDYIPQTKLVDYLNENLQPGDLLVMDTMSFCFALPDIMLPQGVQLLTQNYYGSIGYALPSTFGATMAVNDLGSDRRIILIEGDGAAQMTIQELSSFLKYKEFLPNMPKIFLINNDGYTVERMIKGPTRSYNDINGEWSWTQLLGVFGDKEQKYHSTALLRNVNEFNKYFEFQRQTDNSKLEFIELIAGKYDCPLRFSEMFCKK

Other exemplary sequences are:

Saccharomyces cerevisiae PDC1 amino acid sequence:

(SEQ ID NO: 54) MSEITLGKYLFERLKQVNVNTVFGLPGDFNLSLLDKIYEVEGMRWAGNANELNAAYAADGYARIKGMSCIITTFGVGELSALNGIAGSYAEHVGVLHVVGVPSISAQAKQLLLHHTLGNGDFTVFHRMSANISETTAMITDIATAPAEIDRCIRTTYVTQRPVYLGLPANLVDLNVPAKLLQTPIDMSLKPNDAESEKEVIDTILALVKDAKNPVILADACCSRHDVKAETKKLIDLTQFPAFVTPMGKGSIDEQHPRYGGVYVGTLSKPEVKEAVESADLILSVGALLSDFNTGSFSYSYKTKNIVEFHSDHMKIRNATFPGVQMKFVLQKLLTTIADAAKGYKPVAVPARTPANAAVPASTPLKQEWMWNQLGNFLQEGDVVIAETGTSAFGINQTTFPNNTYGISQVLWGSIGFTTGATLGAAFAAEEIDPKKRVILFIGDGSLQLTVQEISTMIRWGLKPYLFVLNNDGYTIEKLIHGPKAQYNEIQGWDHLSLLPTFGAKDYETHRVATTGEWDKLTQDKSFNDNSKIRMIEIMLPVFDAPQNLVEQAKLTAATNAKQ

Pichia stipitis PDC1 (Ps) amino acid sequence:

(SEQ ID NO: 55) MAEVSLGRYLFERLYQLQVQTIFGVPGDFNLSLLDKIYEVEDAHGKNSFRWAGNANELNASYAADGYSRVKRLGCLVTTFGVGELSALNGIAGSYAEHVGLLHVVGVPSISSQAKQLLLHHTLGNGDFTVFHRMSNNISQTTAFISDINSAPAEIDRCIREAYVKQRPVYIGLPANLVDLNVPASLLESPINLSLEKNDPEAQDEVIDSVLDLIKKSLNPIILVDACASRHDCKAEVTQLIEQTQFPVFVTPMGKGTVDEGGVDGELLEDDPHLIAKVAARLSAGKNAASRFGGVYVGTLSKPEVKDAVESADLILSVGALLSDFNTGSFSYSYRTKNIVEFHSDYTKIRQATFPGVQMKEALQELNKKVSSAASHYEVKPVPKIKLANTPATREVKLTQEWLWTRVSSWFREGDIIITETGTSSFGIVQSRFPNNTIGISQVLWGSIGFSVGATLGAAMAAQELDPNKRTILFVGDGSLQLTVQEISTMIRWGTTPYLFVLNNDGYTIERLIHGVNASYNDIQPWQNLEILPTFSAKNYDAVRISNIGEAEDILKDKEFGKNSKIRLIEVMLPRLDAPSNLAKQAAI TAATNAEA

Saccharomyces cerevisiae PDC5 amino acid sequence:

(SEQ ID NO: 56) MSEITLGKYLFERLSQVNCNTVFGLPGDFNLSLLDKLYEVKGMRWAGNANELNAAYAADGYARIKGMSCIITTFGVGELSALNGIAGSYAEHVGVLHVVGVPSISSQAKQLLLHHTLGNGDFTVFHRMSANISETTAMITDIANAPAEIDRCIRTTYTTQRPVYLGLPANLVDLNVPAKLLETPIDLSLKPNDAEAEAEVVRTVVELIKDAKNPVILADACASRHDVKAETKKLMDLTQFPVYVTPMGKGAIDEQHPRYGGVYVGTLSRPEVKKAVESADLILSIGALLSDFNTGSFSYSYKTKNIVEFHSDHIKIRNATFPGVQMKFALQKLLDAIPEVVKDYKPVAVPARVPITKSTPANTPMKQEWMWNHLGNFLREGDIVIAETGTSAFGINQTTFPTDVYAIVQVLWGSIGFTVGALLGATMAAEELDPKKRVILFIGDGSLQLTVQEISTMIRWGLKPYIFVLNNNGYTIEKLIHGPHAEYNEIQGWDHLALLPTFGARNYETHRVATTGEWEKLTQDKDFQDNSKIRMIEVMLPVFDAPQNLVKQAQLTAATNAKQ

Saccharomyces cerevisiae PDC6 amino acid sequence:

(SEQ ID NO: 57) MSEITLGKYLFERLKQVNVNTIFGLPGDFNLSLLDKIYEVDGLRWAGNANELNAAYAADGYARIKGLSVLVTTFGVGELSALNGIAGSYAEHVGVLHVVGVPSISAQAKQLLLHHTLGNGDFTVFHRMSANISETTSMITDIATAPSEIDRLIRTTFITQRPSYLGLPANLVDLKVPGSLLEKPIDLSLKPNDPEAEKEVIDTVLELIQNSKNPVILSDACASRHNVKKETQKLIDLTQFPAFVTPLGKGSIDEQHPRYGGVYVGTLSKQDVKQAVESADLILSVGALLSDFNTGSFSYSYKTKNVVEFHSDYVKVKNATFLGVQMKFALQNLLKVIPDVVKGYKSVPVPTKTPANKGVPASTPLKQEWLWNELSKFLQEGDVIISETGTSAFGINQTIFPKDAYGISQVLWGSIGFTTGATLGAAFAAEEIDPNKRVILFIGDGSLQLTVQEISTMIRWGLKPYLFVLNNDGYTIEKLIHGPHAEYNEIQTWDHLALLPAFGAKKYENHKIATTGEWDALTTDSEFQKNSVIRLIELKLPVFDAPESLIKQAQLTAATNAKQ

Saccharomyces cerevisiae THI3 amino acid sequence:

(SEQ ID NO: 58) MNSSYTQRYALPKCIAISDYLFHRLNQLNIHTIFGLSGEFSMPLLDKLYNIPNLRWAGNSNELNAAYAADGYSRLKGLGCLITTFGVGELSAINGVAGSYAEHVGILHIVGMPPTSAQTKQLLLHHTLGNGDFTVFHRIASDVACYTTLIIDSELCADEVDKCIKKAWIEQRPVYMGMPVNQVNLPIESARLNTPLDLQLHKNDPDVEKEVISRILSFIYKSQNPAIIVDACTSRQNLIEETKELCNRLKFPVFVTPMGKGTVNETDPQFGGVFTGSISAPEVREVVDFADFIIVIGCMLSEFSTSTFHFQYKTKNCALLYSTSVKLKNATYPDLSIKLLLQKILANLDESKLSYQPSEQPSMMVPRPYPAGNVLLRQEWVWNEISHWFQPGDIIITETGASAFGVNQTRFPVNTLGISQALWGSVGYTMGACLGAEFAVQEINKDKFPATKHRVILFMGDGAFQLTVQELSTIVKWGLTPYIFVMNNQGYSVDRFLHHRSDASYYDIQPWNYLGLLRVFGCTNYETKKIITVGEFRSMISDPNFATNDKIRMIEIMLPPRDVPQALLDRWVVEKEQSKQVQEENENS SAVNTPTPEFQPLLKKNQVGY

Saccharomyces cerevisiae ARO10 amino acid sequence:

(SEQ ID NO: 59) MAPVTIEKFVNQEERHLVSNRSATIPFGEYIFKRLLSIDTKSVFGVPGDFNLSLLEYLYSPSVESAGLRWVGTCNELNAAYAADGYSRYSNKIGCLITTYGVGELSALNGIAGSFAENVKVLHIVGVAKSIDSRSSNFSDRNLHHLVPQLHDSNFKGPNHKVYHDMVKDRVACSVAYLEDIETACDQVDNVIRDIYKYSKPGYIFVPADFADMSVTCDNLVNVPRISQQDCIVYPSENQLSDIINKITSWIYSSKTPAILGDVLTDRYGVSNFLNKLICKTGIWNFSTVMGKSVIDESNPTYMGQYNGKEGLKQVYEHFELCDLVLHFGVDINEINNGHYTFTYKPNAKIIQFHPNYIRLVDTRQGNEQMFKGINFAPILKELYKRIDVSKLSLQYDSNVTQYTNETMRLEDPTNGQSSIITQVHLQKTMPKFLNPGDVVVCETGSFQFSVRDFAFPSQLKYISQGFFLSIGMALPAALGVGIAMQDHSNAHINGGNVKEDYKPRLILFEGDGAAQMTIQELSTILKCNIPLEVIIWNNNGYTIERAIMGPTRSYNDVMSWKWTKLFEAFGDFDGKYTNSTLIQCPSKLALKLEELKNSNKRSGIELLEVKLGELDFPEQLKCMVEAAALKRNKK

Mycobacterium Kdc(Mt) amino acid sequence:

(SEQ ID NO: 60) MTPQKSDACSDPVYTVGDYLLDRLAELGVSEIFGVPGDYNLQFLDHIVAHPTIRWVGSANELNAGYAADGYGRLRGMSAVVTTFGVGELSVTNAIAGSYAEHVPVVHIVGGPTKDAQGTRRALHHSLGDGDFEHFLRISREITCAQANLMPATAGREIDRVLSEVREQKRPGYILLSSDVARFPTEPPAAPLPRYPGGTSPRALSLFTKAAIELIADHQLTVLADLLVHRLQAVKELEALLAADVVPHATLMWGKSLLDESSPNFLGIYAGAASAERVRAAIEGAPVLVTAGVVFTDMVSGFFSQRIDPARTIDIGQYQSSVADQVFAPLEMSAALQALATILTGRGISSPPVVPPPAEPPPAMPARDEPLTQQMVWDRVCSALTPGNVVLADQGTSFYGMADHRLPQGVTFIGQPLWGSIGYTLPAAVGAAVAHPDRRTVLLIGDGAAQLTVQELGTFSREGLSPVIVVVNNDGYTVERAIHGETAPYNDIVSWNWTELPSALGVTNHLAFRAQTYGQLDDALTVAAARRDRMVLVEV VLPRLEIPRLLGQLVGSMAPQ

Lactococcus lactis KdcA(L1) amino acid sequence:

(SEQ ID NO: 61) MYTVGDYLLDRLHELGIEEIFGVPGDYNLQFLDQIISREDMKWIGNANELNASYMADGYARTKKAAAFLTTFGVGELSAINGLAGSYAENLPVVEIVGSPTSKVQNDGKFVHHTLADGDFKHFMKMHEPVTAARTLLTAENATYEIDRVLSQLLKERKPVYINLPVDVAAAKAEKPALSLEKESSTTNTTEQVILSKIEESLKNAQKPVVIAGHEVISFGLEKTVTQFVSETKLPITTLNFGKSAVDESLPSFLGIYNGKLSEISLKNFVESADFILMLGVKLTDSSTGAFTHHLDENKMISLNIDEGIIFNKVVEDFDFRAVVSSLSELKGIEYEGQYIDKQYEEFIPSSAPLSQDRLWQAVESLTQSNETIVAEQGTSFFGASTIFLKSNSRFIGQPLWGSIGYTFPAALGSQIADKESRHLLFIGDGSLQLTVQELGLSIREKLNPICFIINNDGYTVEREIHGPTQSYNDIPMWNYSKLPETFGATEDRVVSKIVRTENEFVSVMKEAQADVNRMYWIELVLEKEDAPKLLKKMG KLFAEQNK

Lactococcus lactis KdcA-S286Y(L1) amino acid sequence:

(SEQ ID NO: 62) MYTVGDYLLDRLHELGIEEIFGVPGDYNLQFLDQIISREDMKWIGNANELNASYMADGYARTKKAAAFLTTFGVGELSAINGLAGSYAENLPVVEIVGSPTSKVQNDGKFVHHTLADGDFKHFMKMHEPVTAARTLLTAENATYEIDRVLSQLLKERKPVYINLPVDVAAAKAEKPALSLEKESSTTNTTEQVILSKIEESLKNAQKPVVIAGHEVISFGLEKTVTQFVSETKLPITTLNFGKSAVDESLPSFLGIYNGKLSEISLKNFVESADFILMLGVKLTDYSTGAFTHHLDENKMISLNIDEGIIFNKVVEDFDFRAVVSSLSELKGIEYEGQYIDKQYEEFIPSSAPLSQDRLWQAVESLTQSNETIVAEQGTSFFGASTIFLKSNSRFIGQPLWGSIGYTFPAALGSQIADKESRHLLFIGDGSLQLTVQELGLSIREKLNPICFIINNDGYTVEREIHGPTQSYNDIPMWNYSKLPETFGATEDRVVSKIVRTENEFVSVMKEAQADVNRMYWIELVLEKEDAPKLLKKMG KLFAEQNK

Lactococcus lactis KdcA-F381W(L1) amino acid sequence:

(SEQ ID NO: 63) MYTVGDYLLDRLHELGIEEIFGVPGDYNLQFLDQIISREDMKWIGNANELNASYMADGYARTKKAAAFLTTFGVGELSAINGLAGSYAENLPVVEIVGSPTSKVQNDGKFVHHTLADGDFKHFMKMHEPVTAARTLLTAENATYEIDRVLSQLLKERKPVYINLPVDVAAAKAEKPALSLEKESSTTNTTEQVILSKIEESLKNAQKPVVIAGHEVISFGLEKTVTQFVSETKLPITTLNFGKSAVDESLPSFLGIYNGKLSEISLKNFVESADFILMLGVKLTDSSTGAFTHHLDENKMISLNIDEGIIFNKVVEDFDFRAVVSSLSELKGIEYEGQYIDKQYEEFIPSSAPLSQDRLWQAVESLTQSNETIVAEQGTSWFGASTIFLKSNSRFIGQPLWGSIGYTFPAALGSQIADKESRHLLFIGDGSLQLTVQELGLSIREKLNPICFIINNDGYTVEREIHGPTQSYNDIPMWNYSKLPETFGATEDRVVSKIVRTENEFVSVMKEAQADVNRMYWIELVLEKEDAPKLLKKMGKLFAEQNK

Lactococcus lactis KdcAS286Y, F381W (L1) amino acid sequence:

(SEQ ID NO: 64) MYTVGDYLLDRLHELGIEEIFGVPGDYNLQFLDQIISREDMKWIGNANELNASYMADGYARTKKAAAFLTTFGVGELSAINGLAGSYAENLPVVEIVGSPTSKVQNDGKFVHHTLADGDFKHFMKMHEPVTAARTLLTAENATYEIDRVLSQLLKERKPVYINLPVDVAAAKAEKPALSLEKESSTTNTTEQVILSKIEESLKNAQKPVVIAGHEVISFGLEKTVTQFVSETKLPITTLNFGKSAVDESLPSFLGIYNGKLSEISLKNFVESADFILMLGVKLTDYSTGAFTHHLDENKMISLNIDEGIIFNKVVEDFDFRAVVSSLSELKGIEYEGQYIDKQYEEFIPSSAPLSQDRLWQAVESLTQSNETIVAEQGTSWFGASTIFLKSNSRFIGQPLWGSIGYTFPAALGSQIADKESRHLLFIGDGSLQLTVQELGLSIREKLNPICFIINNDGYTVEREIHGPTQSYNDIPMWNYSKLPETFGATEDRVVSKIVRTENEFVSVMKEAQADVNRMYWIELVLEKEDAPKLLKKMGKLFAEQNK

Pichia stipitis PDC2 (Ps) amino acid sequence:

(SEQ ID NO: 65) MVSTYPESEVTLGRYLFERLHQLKVDTIFGLPGDFNLSLLDKVYEVPDMRWAGNANELNAAYAADGYSRIKGLSCLVTTFGVGELSALNGVGGAYAEHVGLLHVVGVPSISSQAKQLLLHHTLGNGDFTVFHRMSNSISQTTAFLSDISIAPGQIDRCIREAYVHQRPVYVGLPANMVDLKVPSSLLETPIDLKLKQNDPEAQEEVVETVLKLVSQATNPIILVDACALRHNCKEEVKQLVDATNFQVFTTPMGKSGISESHPRFGGVYVGTMSSPQVKKAVENADLILSVGSLLSDFNTGSFSYSYKTKNVVEFHSDYMKIRQATFPGVQMKEALQQLIKRVSSYINPSYIPTRVPKRKQPLKAPSEAPLTQEYLWSKVSGWFREGDIIVTETGTSAFGIIQSHFPSNTIGISQVLWGSIGFTVGATVGAAMAAQEIDPSRRVILFVGDGSLQLTVQEISTLCKWDCNNTYLYVLNNDGYTIERLIHGKSASYNDIQPWNHLSLLRLFNAKKYQNVRVSTAGELDSLFSDKKFASPDRIRMIEVMLSRL DAPANLVAQAKLSERVNLEN

In particular embodiments of the invention, polypeptide catalyzing theconversion of 2-methylbutanal to 2-methylbutanol is derived from ayeast. An example of a suitable source for this enzyme is the genusSaccharomyces, a preferred source is Saccharomyces cerevisiae.

A specific example of a suitable sequence is:

Saccharomyces cerevisiae ADH6 amino acid sequence:

(SEQ ID NO: 66) MSYPEKFEGIAIQSHEDWKNPKKTKYDPKPFYDHDIDIKIEACGVCGSDIHCAAGHWGNMKMPLVVGHEIVGKVVKLGPKSNSGLKVGQRVGVGAQVFSCLECDRCKNDNEPYCTKFVTTYSQPYEDGYVSQGGYANYVRVHEHFVVPIPENIPSHLAAPLLCGGLTVYSPLVRNGCGPGKKVGIVGLGGIGSMGTLISKAMGAETYVISRSSRKREDAMKMGADHYIATLEEGDWGEKYFDTFDLIVVCASSLTDIDFNIMPKAMKVGGRIVSISIPEQHEMLSLKPYGLKAVSISYSALGSIKELNQLLKLVSEKDIKIWVETLPVGEAGVHEAFERMEKGDVRYRFT LVGYDKEFSD

Other exemplary sequences are:

Saccharomyces cerevisiae ADH1 amino acid sequence:

(SEQ ID NO: 67) MSIPETQKGVIFYESHGKLEYKDIPVPKPKANELLINVKYSGVCHTDLHAWHGDWPLPVKLPLVGGHEGAGVVVGMGENVKGWKIGDYAGIKWLNGSCMACEYCELGNESNCPHADLSGYTHDGSFQQYATADAVQAAHIPQGTDLAQVAPILCAGITVYKALKSANLMAGHWVAISGAAGGLGSLAVQYAKAMGYRVLGIDGGEGKEELFRSIGGEVFIDFTKEKDIVGAVLKATDGGAHGVINVSVSEAAIEASTRYVRANGTTVLVGMPAGAKCCSDVFNQVVKSISIVGSYVGNRADTREALDFFARGLVKSPIKVVGLSTLPEIYEKMEKGQIVGRYVVDTSK

Saccharomyces cerevisiae ADH2 amino acid sequence:

(SEQ ID NO: 68) MSIPETQKAIIFYESNGKLEHKDIPVPKPKPNELLINVKYSGVCHTDLHAWHGDWPLPTKLPLVGGHEGAGVVVGMGENVKGWKIGDYAGIKWLNGSCMACEYCELGNESNCPHADLSGYTHDGSFQEYATADAVQAAHIPQGTDLAEVAPILCAGITVYKALKSANLRAGHWAAISGAAGGLGSLAVQYAKAMGYRVLGIDGGPGKEELFTSLGGEVFIDFTKEKDIVSAVVKATNGGAHGIINVSVSEAAIEASTRYCRANGTVVLVGLPAGAKCSSDVFNHVVKSISIVGSYVGNRADTREALDFFARGLVKSPIKVVGLSSLPEIYEKMEKGQIAGRYVVDTSK

Saccharomyces cerevisiae ADH3 amino acid sequence:

(SEQ ID NO: 69) MLRTSTLFTRRVQPSLFSRNILRLQSTAAIPKTQKGVIFYENKGKLHYKDIPVPEPKPNEILINVKYSGVCHTDLHAWHGDWPLPVKLPLVGGHEGAGVVVKLGSNVKGWKVGDLAGIKWLNGSCMTCEFCESGHESNCPDADLSGYTHDGSFQQFATADAIQAAKIQQGTDLAEVAPILCAGVTVYKALKEADLKAGDWVAISGAAGGLGSLAVQYATAMGYRVLGIDAGEEKEKLFKKLGGEVFIDFTKTKNMVSDIQEATKGGPHGVINVSVSEAAISLSTEYVRPCGTVVLVGLPANAYVKSEVFSHVVKSINIKGSYVGNRADTREALDFFSRGLIKSPIKIVGLSELPKVYDLMEKGKILGRYVVDTSK

Pichia stipitis ADH3 (Ps) amino acid sequence:

(SEQ ID NO: 70) MSKSTSTTVPAKFSGFAVDKPENWNKAKLVQYDPKPFKPYDITIKVICCGVCGSDCHTVLGSWGPLNRDDLVVGHEIVGEVIEIGSEVTNHKLGDIVAVGAQSDSCGECELCENNNEQYCRDGIAATYNFPNKRCGGYVTQGGYASHLRVNSYFAASVPKNLDVHYAAPLLCGGLTVYSPIVRHGGYDLKDKRIGIVGIGGLGSMAIQIANALGAKEVVAFSRTSDKKEDALKLGASRIIATKEDPDWSKSNAATFDIILNCASFGKGVNFDSFFGALKLGGKYVNVSAPPSDELISLSPRNLIFGGFSIVGSVIGSMKEANELLKLYADNNLAPWIEKVPISEEGVHTVMNRINVSDVKYRFVLTDYDKAFNN

Pichia stipitis ADH6 (Ps) amino acid sequence:

(SEQ ID NO: 71) MTTSRTVPEKFSGFGVDKAENWNKARLVRFDPKPLMPYDITIKVIACAVCGSDCHTVTGNFGPINRDDLVVGHEIVGEVIEVGPEVTKHKLGDVVAIGAQSDSCGECNRCKSNNEQYCQKGTVGTYNSLSKKCGGYITQGGYASHVRVNSHFAARVPANLDVHHAAPLLCGGLTVYSPIVRHAGYDLKEKVIGIVGIGGLGSMAIQIAKALGAKEVVAFSRSSSKKEDAFKMGASKYIATKEDTEWANSNLDTFDMILNCASFGKGVDYDSFIRTLKLGGKYVTVSAPPADESITIAPFNLLIGGGIIAGSGIGSMKEADELLKLYADNNLAPWIEKVPISEEGVHKVMNRISVGDVRYRFVLTDFDQAFDSKW

Saccharomyces cerevisiae ADH7 amino acid sequence:

(SEQ ID NO: 72) MLYPEKFQGIGISNAKDWKHPKLVSFDPKPFGDHDVDVEIEACGICGSDFHIAVGNWGPVPENQILGHEIIGRVVKVGSKCHTGVKIGDRVGVGAQALACFECERCKSDNEQYCTNDHVLTMWTPYKDGYISQGGFASHVRLHEHFAIQIPENIPSPLAAPLLCGGITVFSPLLRNGCGPGKRVGIVGIGGIGHMGILLAKAMGAEVYAFSRGHSKREDSMKLGADHYIAMLEDKGWTEQYSNALDLLVVCSSSLSKVNFDSIVKIMKIGGSIVSIAAPEVNEKLVLKPLGLMGVSISSSAIGSRKEIEQLLKLVSEKNVKIWVEKLPISEEGVSHAFTRMESGDVKYRF TLVDYDKKFHK

Pichia stipitis ADH7 (Ps) amino acid sequence:

(SEQ ID NO: 73) MGYPDTFQGFAVNDTSKWSEVEKMDFKPKTFGPLDIDIKIKACGVCGSDVHTVTGGWDQPRLPVIVGHEIVGEVVKVGDNVSSFKIGDRVGMGAQAWACLECDVCKNGDEIYCPKWVDTYNDVYPDGSLAYGGYSSHVRVHEHFAFPIPEALSTEGVAPMLCAGITTYSPLVRNGAGPGKKVGVVGVGGLGHFAIMWARALGCEVYVFSRSLSKKDDAIKLGADHYIATGEENWNEPYKYKLDLILSTANSNSGFDMGAYLSTLRVHGKYIALGLPEDDFKVSPESLLKNGCFVGSSHLGNRQEMIDMLNLAAEKGIEAWYEAVPIGKQGIKEALERCQSGKVKYRFTLT DYEKQFE

Saccharomyces cerevisiae GRE2 amino acid sequence:

(SEQ ID NO: 74) MSVFVSGANGFIAQHIVDLLLKEDYKVIGSARSQEKAENLTEAFGNNPKFSMEVVPDISKLDAFDHVFQKHGKDIKIVLHTASPFCFDITDSERDLLIPAVNGVKGILHSIKKYAADSVERVVLTSSYAAVFDMAKENDKSLTFNEESWNPATWESCQSDPVNAYCGSKKFAEKAAWEFLEENRDSVKFELTAVNPVYVFGPQMFDKDVKKHLNTSCELVNSLMHLSPEDKIPELFGGYIDVRDVAKAHLVAFQKRETIGQRLIVSEARFTMQDVLDILNEDFPVLKGNIPVGKPGSGATHNTLGATLDNKKSKKLLGFKFRNLKETIDDTASQILKFEGRI

Pichia stipitis GRE2 (Ps) amino acid sequence:

(SEQ ID NO: 75) MTSVFVSGATGFIAQHVVKDLLAKNYTVIGSVRSASKGDHLAELLGSKKFSYEVVEDIEKEGAFDAALEKHPEVSVFLHTASPFHFKATDNEKELLLPAVNGTKNAFRAIQLHGKNVTNVVLTSSYAAVGTASKDANKDEVINEESWNEITWEEALKDPVSGYRGSKTFAEKAAWEFLKENNPKFVLSVVNPTFVFGPQAFDSEVKDSLNTSSEVINALLKSGANGVVPPVKGGFVDVRDVSSAHITAFEKEAAYGQRLILNSTRFTAQEIVDILNKRFPELVGKIPVGEPGTGPSLRANNATIDNTKTKKILGVSEFIGLEKSVVDSVSQILRTRK

Saccharomyces cerevisiae SFA1 amino acid sequence:

(SEQ ID NO: 76) MSAATVGKPIKCIAAVAYDAKKPLSVEEITVDAPKAHEVRIKIEYTAVCHTDAYTLSGSDPEGLFPCVLGHEGAGIVESVGDDVITVKPGDHVIALYTAECGKCKFCTSGKTNLCGAVRATQGKGVMPDGTTRFHNAKGEDIYHFMGCSTFSEYTVVADVSVVAIDPKAPLDAACLLGCGVTTGFGAALKTANVQKGDTVAVFGCGTVGLSVIQGAKLRGASKIIAIDINNKKKQYCSQFGATDFVNPKEDLAKDQTIVEKLIEMTDGGLDFTFDCTGNTKIMRDALEACHKGWGQSIIIGVAAAGEEISTRPFQLVTGRVWKGSAFGGIKGRSEMGGLIKDYQKGALKVEEFITHRRPFKEINQAFEDLHNGDCLRTVLKSDEIK

Saccharomyces cerevisiae YPR1 amino acid sequence:

(SEQ ID NO: 77) MPATLKNSSATLKLNTGASIPVLGFGTWRSVDNNGYHSVIAALKAGYRHIDAAAIYLNEEEVGRAIKDSGVPREEIFITTKLWGTEQRDPEAALNKSLKRLGLDYVDLYLMHWPVPLKTDRVTDGNVLCIPTLEDGTVDIDTKEWNFIKTWELMQELPKTGKTKAVGVSNFSINNIKELLESPNNKVVPATNQIEIHPLLPQDELIAFCKEKGIVVEAYSPFGSANAPLLKEQAIIDMAKKHGVEPAQLIISWSIQRGYVVLAKSVNPERIVSNFKIFTLPEDDFKTISNLSKVHGTKRV VDMKWGSFPIFQ

Mycobacterium ADH1(Mt) amino acid sequence:

(SEQ ID NO: 78) MPAPDTIRPHSTSIRAAVFDGTISVEPVDLADPRPGEVRVKIAAAGVCHSDLHVTTGAWDVPAPVVLGHEGSGVVTAVGEGVDDLEPGDHVVLSWVPGCGECRYCKAGRPAQCSLVASVVAVKGTLYDGTTRLSNERGTVHHYLGVSSYAEQVVVPRNGAIKVRKDAPLEDIAIVGCAIATGVGAVRNTAGVEPGSTVAVIGCGGVGLACVQGARLAGASRIVAVDVVAEKLELARKLGATDAVDASATDDVVAAMREVLPDGYDYVFDAIGKIATTEQAIAALGLGGAAVIVGLPPQGERASFDPLTLAEADQRILGSNYGSAVPERDIPALVDEVMAGNLDLASMISGRRPLEEAAAALDDLAAGHALRQLLIPSA

Mycobacterium ADHs(Mt) amino acid sequence:

(SEQ ID NO: 79) MRAVDGFPGRGAVITGGASGIGLATGTEFARRGARVVLGDVDKPGLRQAVNHLRAEGFDVHSVMCDVRHREEVTHLADEAFRLLGHVDVVFSNAGIVVGGPIVEMTHDDWRWVIDVDLWGSIHTVEAFLPRLLEQGTGGHVVFTASFAGLVPNAGLGAYGVAKYGVVGLAETLAREVTADGIGVSVLCPMVVETNLVANSERIRGAACAQSSTTGSPGPLPLQDDNLGVDDIAQLTADAILANRLYVLPHAASRASIRRRFERIDRTFDEQAAEGWRH

Mycobacterium dhb(Mt) amino acid sequence:

(SEQ ID NO: 80) MKTKGALIWEFNQPWSVEEIEIGDPRKDEVKIQMEAAGMCRSDHHLVTGDIPMAGFPVLGGHEGAGIVTEVGPGVDDFAPGDHVVLAFIPSCGKCPSCQAGMRNLCDLGAGLLAGESVTDGSFRIQARGQNVYPMTLLGTFSPYMVVHRSSVVKIDPSVPFEVACLVGCGVTTGYGSAVRTADVRPGDDVAIVGLGGVGMAALQGAVSAGARYVFAVEPVEWKRDQALKFGATHVYPDINAALMGIAEVTYGLMAQKVIITVGKLDGADVDSYLTITAKGGTCVLTAIGSLVDTQVTLNLAMLTLLQKNIQGTIFGGGNPHYDIPKLLSMYKAGKLNLDDMVTTAYKLEQINDGYQDMLNGKNIRGVIRYTDDDR

Equus caballus ADHE(Horse) amino acid sequence:

(SEQ ID NO: 81) MSTAGKVIKCKAAVLWEEKKPFSIEEVEVAPPKAHEVRIKMVATGICRSDDHVVSGTLVTPLPVIAGHEAAGIVESIGEGVTTVRPGDKVIPLFTPQCGKCRVCKHPEGNFCLKNDLSMPRGTMQDGTSRFTCRGKPIHHFLGTSTFSQYTVVDEISVAKIDAASPLEKVCLIGCGFSTGYGSAVKVAKVTQGSTCAVFGLGGVGLSVIMGCKAAGAARIIGVDINKDKFAKAKEVGATECVNPQDYKKPIQEVLTEMSNGGVDFSFEVIGRLDTMVTALSCCQEAYGVSVIVGVPPDSQNLSMNPMLLLSGRTWKGAIFGGFKSKDSVPKLVADFMAKKFALDPLITHVLPFEKINEGFDLLRSGESIRTILTF

Steps a) to i) discussed above, converting malate to threonine, can beachieved through an alternative pathway, which is illustrated in FIG. 2.This pathway describes the conversion of pyruvate (and acetal CoA) tocitramalate (step a′), citramalate to erythro-beta-methyl-D-malate(2-methylfumaric acid) (step b′), and erythro-beta-methyl-D-malate to2-oxobutanoate (step c′). The product of step c′ enters the pathwayshown in FIG. 1 at step j.

The designations provide examples of enzymes that catalyze particularreactions in the overall pathway. For example, citramalate synthase isan example of a designation for the enzyme that catalyzes step a′.Because enzymatic nomenclature various between organisms, it should benoted that the names provided below are merely illustrative of a classof enzymes that catalyze the particular steps of the pathway. Theenzymes contemplated for use with the invention are those that catalyzethe reactions illustrated and are not limited to the enzymatic namesprovided.

The designations in the figure are:

Step a′) citramalate synthase [EC 4.1.3.22] or 2-isopropylmalatesynthase [EC 2.3.3.13];

Step b′) an isopropylmalate isomerase [EC 4.2.1.33]; and

Step c′) isopropylmalate dehydrogenase [EC 1.1.1.85].

A fourth aspect of the invention provides a recombinant microorganismcomprising at least one DNA molecule, wherein said at least one DNAmolecule encodes at least two polypeptides that catalyze a substrate toproduct conversion selected from the group consisting of steps a′)through c′), and wherein said recombinant microorganism produces2-methylbutanol.

In particular embodiments of the invention, the polypeptide catalyzingthe conversion of pyruvate (and acetal CoA) to citramalate is derivedfrom a bacterium. An example of a suitable source for this enzyme is thegenus Thermotoga, a preferred source is Thermotoga maritima.

A specific example of a suitable sequence is:

Thermatoga maritima leuA amino acid sequence:

(SEQ ID NO: 82) MSIKVYDTTLRDGAQAFGVSFSLEDKIRIAEALDDLGVHYLEGGWPGSNPKDIAFFEAVKGMNFKNLKVAAFSSTRRPDVKIEEDANIQTLIKAETPVYTIFGKSWDLHVEKALRTTLEENLKMIYDTVSYLKRFADEVIYDAEHFFDGYKANREYALKTLKVAEEAGADCLVLADTNGGTLPHEIEEIIEDVKKHVKAPLGIHAHNDSDVAVANTLAAVRKGAVHVQGTINGLGERCGNANLCSVIPNLVLKMGLEVIPKENLKKLFDVAHLVAELSGRPHIENMPYVGDYAFAHKGGVHVSAIKRDPRTYEHIDPELVGNRRIISISELSGKSNVLEKIKEMGFEIDESSPKVREILKKIKELEAQGYHFEGAEASFELLVRDMLGKRKKYFEFLGFTVMTIKNRDEESFSEATVKVRVPDEVAKRLGHDEPFEHTAAEGEGPVEALDRAVRKALEKFYPSLKDTKLTDYKVRILNEQAGTKATTRVLIESSDGKRRWGTVGVSPNIIEASWTALLESLEYKLHKDEEEMRNDEEN

Other exemplary sequences are:

Thermatoga maritima leuA truncated (i) amino acid sequence:

(SEQ ID NO: 83) MSIKVYDTTLRDGAQAFGVSFSLEDKIRIAEALDDLGVHYLEGGWPGSNPKDIAFFEAVKGMNFKNLKVAAFSSTRRPDVKIEEDANIQTLIKAETPVYTIFGKSWDLHVEKALRTTLEENLKMIYDTVSYLKRFADEVIYDAEHFFDGYKANREYALKTLKVAEEAGADCLVLADTNGGTLPHEIEEIIEDVKKHVKAPLGIHAHNDSDVAVANTLAAVRKGAVHVQGTINGLGERCGNANLCSVIPNLVLKMGLEVIPKENLKKLFDVAHLVAELSGRPHIENMPYVGDYAFAHKGGVHVSAIKRDPRTYEHIDPELVGNRRIISISELSGKSNVLEKIKEMGFEIDESSPKVREILKKIKELEAQGYHFEGAEASFELL

Thermatoga maritima leuA truncated (ii) amino acid sequence:

(SEQ ID NO: 84) MSIKVYDTTLRDGAQAFGVSFSLEDKIRIAEALDDLGVHYLEGGWPGSNPKDIAFFEAVKGMNFKNLKVAAFSSTRRPDVKIEEDANIQTLIKAETPVYTIFGKSWDLHVEKALRTTLEENLKMIYDTVSYLKRFADEVIYDAEHFFDGYKANREYALKTLKVAEEAGADCLVLADTNGGTLPHEIEEIIEDVKKHVKAPLGIHAHNDSDVAVANTLAAVRKGAVHVQGTINGLGERCGNANLCSVIPNLVLKMGLEVIPKENLKKLFDVAHLVAELSGRPHIENMPYVGDYAFAHKGGV HVSAIKRDPRTYEHID

Synechocystis leuA amino acid sequence:

(SEQ ID NO: 85) MATKKTSLWLYDTTLRDGAQREGISLSLTDKLTIARRLDQLGIPFIEGGWPGANPKDVQFFWQLQEEPLEQAEIVAFCSTRRPHKAVETDKMLQAILSAGTRWVTIFGKSWDLHVLEGLQTSLAENLAMISDTIAYLRSQGRRVIYDAEHWFDGYRANPDYALATLATAQQAGAEWLVMCDTNGGTLPGQISEITTKVRRSLGLDGQSDRQPQLGIHAHNDSGTAVANSLLAVEAGATMVQGTINGYGERCGNANLCTLIPNLQLKLDYDCIEPEKLAHLTSTSRLISEIVNLAPDDHAPFVGRSAFAHKGGIHVSAVQRNPFTYEHIAPNLVGNERRIVVSEQAGLSNVLSKAELFGIALDRQNPACRTILATLKDLEQQGYQFEAAEASFELLMRQAMGDRQPLFLVQGFQVHCDLLTPAENPAYRNALATVKVTVNGQNILEVAEGNGPVSALDQALRKALTRFYPQIADFHLTDYKVRILDGGAGTSAKTRVLVESSNGDRRWTTVGVSGNILEASYQAVVEGIEYGLRLLTCGLTNQEAISS

Geobacter sulfurreducens cimA amino acid sequence:

(SEQ ID NO: 86) MSLVKLYDTTLRDGTQAEDISFLVEDKIRIAHKLDEIGIHYIEGGWPGSNPKDVAFFKDIKKEKLSQAKIAAFGSTRRAKVTPDKDHNLKTLIQAEPDVCTIFGKTWDFHVHEALRISLEENLELIFDSLEYLKANVPEVFYDAEHFFDGYKANPDYAIKTLKAAQDAKADCIVLCDTNGGTMPFELVEIIREVRKHITAPLGIHTHNDSECAVANSLHAVSEGIVQVQGTINGFGERCGNANLCSIIPALKLKMKRECIGDDQLRKLRDLSRFVYELANLSPNKHQAYVGNSAFAHKGGVHVSAIQRHPETYEHLRPELVGNMTRVLVSDLSGRSNILAKAEEFNIKMDSKDPVTLEILENIKEMENRGYQFEGAEASFELLMKRALGTHRKFFSVIGFRVIDEKRHEDQKPLSEATIMVKVGGKIEHTAAEGNGPVNALDNALRKALEKFYPRLKEVKLLDYKVRVLPAGQGTASSIRVLIESGDKESRWGTVGVSENIVDASYQALLDSVEYKLHKSEEIEGSKK

In particular embodiments of the invention, the polypeptide catalyzingthe conversion of citramalate to erythro-beta-methyl-D-malate(2-methylfumaric acid) is derived from a bacterium. An example of asuitable source for this enzyme is the genus Methanococcus, a preferredsource is Methanococcus jannaschii.

Specific examples of suitable sequences are:

Methanococcus jannaschii leuC amino acid sequence:

(SEQ ID NO: 87) MGMTIVEKILAKASGKKEVSPGDIVMANIDVAMVHDITGPLTVNTLKEYGIEKVWNPEKIVILFDHQVPADSIKAAENHILMRKFVKEQGIKYFYDIREGVCHQVLPEKGHVAPGEVVVGADSHTCTHGAFGAFATGIGSTDMAHVFATGKLWFKVPETIYFNITGDLQPYVTSKDVILSIIGEVGVDGATYKACQFGGETVKKMSIASRMTMTNMAIEMGGKTGIIEPDEKTIQYVKEAMKKHGTERPFEVIKGDEDAEFAEVYEIEADKIEPVFACPHNVDNVKQAREVAGKPIDQVFIGSCTNGRLEDLRMAIKIIEKHGGIADDVRVVVTPASREEYLKALKEGIIEKFLKYGCVVTNPSCSACMGSLYGVLGPGEVCVSTSNRNFRGRQGSLEAEIYLASPITAAACAVKGELVDPRDL

Methanococcus jannaschii leuD amino acid sequence:

(SEQ ID NO: 88) MRSIIKGRVWKFGNNVDTDAILPARYLVYTKPEELAQFVMTGADPDFPKKVKPGDIIVGGKNFGCGSSREHAPLGLKGAGISCVIAESFARIFYRNAINVGLPLIECKGISEKVNEGDELEVNLETGEIKNLTTGEVLKGQKLPEFMMEI LEAGGLMPYLKKKMAESQ

In particular embodiments of the invention, the polypeptide catalyzingthe conversion of erythro-beta-methyl-D-malate to 2-oxobutanoate isderived from a bacterium. An example of a suitable source for thisenzyme is the genus Methanococcus, a preferred source is Methanococcusjannaschii.

A specific example of a suitable sequence is: Methanococcus jannaschiileuB amino acid sequence:

(SEQ ID NO: 89) MHKICVIEGDGIGKEVVPATIQVLEATGLPFEFVYAEAGDEVYKRTGKALPEETIETALDCDAVLFGAAGETAADVIVKLRHILDTYANIRPVKAYKGVKCLRPDIDYVIVRENTEGLYKGIEAEIDEGITIATRVITEKACERIFRFAFNLARERKKMGKEGKVTCAHKANVLKLTDGLFKKIFYKVAEEYDDIKAEDYYIDAMNMYIITKPQVFDVVVTSNLFGDILSDGAAGTVGGLGLAPSANIGDEHGLFEPVHGSAPDIAGKKIANPTATILSAVLMLRYLGEYEAADKVEKALEEVLALGLTTPDLGGNLNTFEMAEEVAKRVREE

Any of the foregoing recombinant microorganisms may further comprise anucleic acid encoding an amino acid biosynthesis regulatory protein.

A specific example of a suitable sequence is:

Saccharomyces cerevisiae GCN4 amino acid sequence:

(SEQ ID NO: 90) MSEYQPSLFALNPMGFSPLDGSKSTNENVSASTSTAKPMVGQLIFDKFIKTEEDPIIKQDTPSNLDFDFALPQTATAPDAKTVLPIPELDDAVVESFFSSSTDSTPMFEYENLEDNSKEWTSLFDNDIPVTTDDVSLADKAIESTEEVSLVPSNLEVSTTSFLPTPVLEDAKLTQTRKVKKPNSVVKKSHHVGKDDESRLDHLGVVAYNRKQRSIPLSPIVPESSDPAALKRARNTEAARRSRARKLQRMKQLEDKVEELLSKNYHLENEVARLKKLVGER

The genes of these pathways are well studied and many examples for eachstep of the pathway are available in the literature. Table 1 belowprovides a number of exemplars.

TABLE 1 List of Recombinant Enzymes Evaluated for the EnhancedProduction of MBO Protein EC# Accession # Enzyme Name [Genus species]GENE Malate to Pyruvate 1.1.1.37 ABN66921 Malate Dehydrogenase [Pichiastipitis] MDH2 (Ps) AAA34766 Malate Dehydrogenase [Saccharomycescerevisiae] MDH2 Pyruvate to Threonine 6.4.1.1 CAA96765 Pyruvatecarboxylase [Saccharomyces cerevisiae] PYC1 ABN68200 Pyruvatecarboxylase [Pichia stipitis] PYC1 (Ps) CAA85182 Pyruvate carboxylase[Saccharomyces cerevisiae] PYC2 ABN68195 Acetyl-CoA transporter [Pichiastipitis] PYC2 (Ps) 2.6.1.1 CAA50451 Aspartate aminotransferase[Saccharomyces AAT1 cerevisiae] EAZ63967 Aspartate aminotransferase[Pichia stipitis] AAT1 (Ps) P23542 Aspartate aminotransferase[Saccharomyces AAT2 cerevisiae] ABN68070 Aspartate aminotransferase[Pichia stipitis] AAT2 (Ps) 2.7.2.4 AAB64587 Aspartate kinase(L-aspartate 4-P-transferase) HOM3^(FBR) [Saccharomyces cerevisiae]EAZ63309 Aspartate kinase (L-aspartate 4-P-transferase) HOM3^(FBR)[Pichia stipitis] (Ps) 1.2.1.11 AAS56024 HOM2 Aspartic betasemi-aldehyde dehydrogenase HOM2 (Sc) [Saccharomyces cerevisiae]ABN66253 HOM2 Aspartic beta semi-aldehyde dehydrogenase HOM2 (Ps)[Pichia stipitis] 1.1.1.3 CAA45787 Homoserine dehydrogenase[Saccharomyces HOM6 cerevisiae] ABN65351 Homoserine dehydrogenase[Pichia stipitis] HOM6 (Ps) 2.7.1.39 AAA35154 Homoserine kinase[Saccharomyces cerevisiae] THR1 ABN68112 Homoserine kinase [Pichiastipitis] THR1 (Ps) NP600410 Homoserine kinase [Corynebacteriumglutamicum] KhsE (Cg) 4.2.99.2 CAA42284 Threonine synthase[Saccharomyces cerevisiae] THR4 ABN67095 Threonine synthase [Pichiastipitis] THR4 (Ps) Threonine to KMV 4.3.1.19 AAB64641 Threoninedeaminase [Saccharomyces cerevisiae] ILV1 AAB64641 Threonine deaminase[Saccharomyces cerevisiae] ILV1^(FBR) ABN67213 Threonine deaminase[Pichia stipitis] ILV1 (Ps)^(FBR) ABN67213 Threonine deaminase [Pichiastipitis] ILV1 (Ps) Δ15 ABN67213 Threonine deaminase [Pichia stipitis]ILV1 (Ps) CAA42403 Catabolic L-serine (L-threonine) deaminase CHA1[Saccharomyces cerevisiae] CAF20464 Threonine dehydratase[Corynebacterium IlvA (Cg) glutamicum] AAB59054 Threonine deaminase[Escherichia coli] IlvA (Ec) 2.2.1.6 CAA89744 Acetolactate synthase[Saccharomyces cerevisiae] ILV2 ABN66585 Acetolactate synthase [Pichiastipitis] ILV2 (Ps) ABN66585 Acetolactate synthase [Pichia stipitis]ILV2 (Ps)Δ26 CAA42350 Acetolactate synthase regulatory subunit ILV6[Saccharomyces cerevisiae] EAZ63909 Acetolactate synthase regulatorysubunit [Pichia ILV6 (Ps) stipitis] BAB98664 Acetolactate synthase 1catalytic subunit IlvB (Cg) [Corynebacterium glutamicum] CAF19975Acetolactate synthase I, small subunit ilvN (Cg) [Corynebacteriumglutamicum] BAE77622 Acetolactate synthase I, large subunit [EscherichiailvB(Ec) coli] BAE77623 Acetolactate synthase I, small subunit[Escherichia ilvN (Ec) coli] AAA67571 Acetolactate synthase II, largesubunit [Escherichia ilvG(Ec) coli] BAE77528 Acetolactate synthase II,small subunit [Escherichia ilvM(Ec) coli] BAB96646 Acetolactate synthaseIII, large subunit [Escherichia ilvl(Ec) coli] BAB96647 Acetolactatesynthase III, thiamin-dependent ilvIH(Ec) [Escherichia coli] 1.1.1.86CAA28643 Acetohydroxyacid reductoisomerase [Saccharomyces ILV5cerevisiae] ABN66666 Mitochondrial ketol-acid reductoisomerase [PichiaILV5 (Ps) stipitis] ABN66666 Mitochondrial ketol-acid reductoisomerase[Pichia ILV5 (Ps) stipitis] Δ40 CAF19976 Ketol-acid reductoisomerase[Corynebacterium IlvC (cg) glutamicum] BAE77523 Ketol-acidreductoisomerase, NAD(P)-binding ilvC (Ec) [Escherichia coli] 4.2.1.9CAA60939 Dihydroxyacid dehydratase [Saccharomyces ILV3 cerevisiae]ABN65237 Dihydroxyacid dehydratase [Pichia stipitis] ILV3 (Ps) ABN65237Dihydroxyacid dehydratase [Pichia stipitis] ILV3 (Ps)Δ34 CAF19971Dihydroxyacid dehydratase [Corynebacterium IlvD (Cg) glutamicum]BAE77526 Dihydroxyacid dehydratase [Escherichia coli] ilvD (Ec) KMV to2MBO 4.1.1.72 CAA97573 Major of three pyruvate decarboxylase isozymesPDC1 [Saccharomyces cerevisiae] EAZ63546 Pyruvate decarboxylase [Pichiastipitis] PDC1 (Ps) CAA97705 Minor isoform of pyruvate decarboxylasePDC5 [Saccharomyces cerevisiae] CAA39398 Minor isoform of pyruvatedecarboxylase PDC6 [Saccharomyces cerevisiae] CAA98646 Probablealpha-ketoisocaproate decarboxylase THI3 [Saccharomyces cerevisiae]AAB64816 Pyruvate decarboxylase [Saccharomyces cerevisiae] ARO10ABN67867 Pyruvate decarboxylase (PDC6) (PDC3) [Pichia PDC 3-6 (Ps)stipitis] O53865 Branched-chain alpha-ketoacid decarboxylase Kdc(Mt)[Mycobacterium] AAS49166 Branched-chain alpha-ketoacid decarboxylaseKdcA(LI) [Lactococcus lactis] AAS49166 Branched-chain alpha-ketoaciddecarboxylase KdcA- [Lactococcus lactis] S286Y(LI) AAS49166Branched-chain alpha-ketoacid decarboxylase KdcA- [Lactococcus lactis]F381W(LI) AAS49166 Branched-chain alpha-ketoacid decarboxylaseKdcAS286Y, [Lactococcus lactis] F381W (LI) EAZ63682 Pyruvatedecarboxylase [Pichia stipitis] PDC2 (Ps) 1.1.1.1 CAA58193 Alcoholdehydrogenase [Saccharomyces cerevisiae] ADH1 AAA34408 Alcoholdehydrogenase [Saccharomyces cerevisiae] ADH2 CAA89229 Alcoholdehydrogenase [Saccharomyces cerevisiae] ADH3 ABN65575 Alcoholdehydrogenase (NADP dependent) [Pichia ADH3 (Ps) stipitis] CAA90836Alcohol dehydrogenase [Saccharomyces cerevisiae] ADH6 EAZ62840NADP-dependent alcohol dehydrogenase [Pichia ADH6 (Ps) stipitis]CAA42237 NADPH-dependent alcohol dehydrogenase ADH7 [Saccharomycescerevisiae] ABN66271 NADPH-dependent alcohol dehydrogenase [Pichia ADH7(Ps) stipitis] CAA88277 NADPH-dependent methylglyoxal reductase GRE2[Saccharomyces cerevisiae] ABN66052 NADPH-dependent methylglyoxalreductase GRE2 GRE2 (Ps) [Pichia stipitis] CAA91578 Bifunctionalenzyme-alcohol dehydrogenase and SFA1 glutathione-dependent formaldehydedehydrogenase activities [Saccharomyces cerevisiae] CAA566862-methylbutyraldehyde reductase [Saccharomyces YPR1 cerevisiae] ABK75278Alcohol dehydrogenase 1 [Mycobacterium] ADH1(Mt) AAK45115 Alcoholdehydrogenase small [Mycobacterium] ADHs(Mt) CAE55322 Zinc-containingalcohol dehydrogenase NAD Adhb(Mt) dependent ADHB [Mycobacterium] P00327Alcohol dehydrogenase-E-isoenzyme [Equus ADHE(Horse) caballus]Citramalate 2.3.3.13 AAD35638 2-isopropylmalate synthase [Thermatogamaritima] leuA 2-isopropylmalate synthase [Thermatoga maritima] leuAtruncated NP442009 2-isopropylmalate synthase [Synechocystis] leuA4.1.3.22 GSU1798 Citramalate synthase [Geobacter sulfurreducens] cimA4.2.1.33 MJ0499 Isopropylmalate isomerase [Methanococcus leuCjannaschii] MJ1277 Isopropylmalate isomerase [Methanococcus leuDjannaschii] 1.1.1.85 MJ0720 Isopropylmalate dehydrogenase [MethanococcusleuB jannaschii] Nitrogen regulation NA P03069 Amino acid biosynthesisregulatory protein GCN4 [Saccharomyces cerevisiae]

FIG. 3 shows a proposed pathway for the generation of 2-MBO withleucine, valine, isoleucine, 2-methyl butyric acid,2,3-dihydroxy-3-methyl valeric acid, isoleucine, 2-ketoisovaleric acid,or 2-methylbutyryl CoA as the starting material. Each step of theenzymatic pathway is provided with a letter designation whichcorresponds to an enzyme.

-   -   Step a) corresponds to the conversion of leucine, to        2-keto-3-methyl valeric acid (KMV),    -   Step b) corresponds to the conversion of valine to        2-keto-3-methyl valeric acid (KMV),    -   Step c) corresponds to the conversion of isoleucine to        2-keto-3-methyl valeric acid (KMV),    -   Step d) corresponds to the conversion of 2-methylbutyryl CoA to        KMV,    -   Step e) corresponds to the conversion of 2,3-dihydroxy-3-methyl        valeric acid to KMV,    -   step f) corresponds to the conversion of isoleucine and        2-ketoisovaleric acid to KMV,    -   Step g) corresponds to the conversion of 2-methylbutyryl CoA to        KMV,    -   Step h) corresponds to the conversion of KMV to        2-methyl-1-butanal, and    -   Step i) corresponds to the conversion of 2-methyl-1-butanal to        2-methyl-1-butanol.

The designations provide examples of enzymes that catalyze particularreactions in the overall pathway. For example, valine-isoleucineamniotransferase is an example of a designation for the enzyme thatcatalyzes the conversion of leucine, valine, and isoleucine to2-keto-3-methyl valeric acid (KMV). Because enzymatic nomenclaturevarious between organisms, it should be noted that the names providedabove are merely illustrative of a class of enzymes that catalyze theparticular steps of the pathway. The enzymes contemplated for use withthe invention are those that catalyze the reactions illustrated and arenot limited to the enzymatic names provided.

The conversion of isoleucine to 2-keto-3-methylvalerate is catalyzed byvaline-isoleucine aminotransferase (EC 2.6.1.32) or branched-chain aminoacid transaminase (EC 2.6.1.42), which may be encoded by, but notlimited to, one or more of the following genes: O14370; P38891; P47176;Q93Y32; P54687; P24288; P54690; Q9GKM4; Q9M439; Q5EA40; O15382; O35855;O19098; Q5REP0; O35854; Q9M401; Q9FYA6; Q9LPM9; P54688; O67733; O29329;P39576; P0AB82; P0AB81; P0AB80; P54689; Q9ZJF1; O26004; Q58414; O27481;O32954; Q10399; O86428; Q1RIJ2; Q92I26; Q4ULR3; O05970; Q9AKE5; P0A1A6;P0A1A5; Q5HIC1; P63512; P99138; Q6GJB4; Q6 GBT3; P63513; Q5HRJ8; Q8CQ78;O86505; P54691; P74921; Q9Y885; and O31461.

The conversion of 2-methylbutyrate to 2-keto-3-methylvalerate iscatalyzed by 2-methylbutyrate decarboxylase, which may be encoded by,but not limited to, genes occurring naturally in anaerobicmicroorganisms.

The conversion of 2,3-dihydroxy-3-methylvalerate to2-keto-3-methylvalerate is catalyzed by dihydroxyacid dehydratase (EC4.2.1.9), which may be encoded by, but not limited to, one or more ofthe following genes:

Q10318; P39522; Q6FCR9; Q5P8J4; Q7WQA2; Q7WC98; Q7W069; Q89LK8; Q394V3;Q8FPX6; Q8TPV2; Q5Z0M2; Q3IJH1; Q475B2; Q98BZ8; Q49Z08; Q6F6Q0; Q5P6F1;Q7WJP7; Q7W497; Q7VUN6; Q89KY5; Q39DS9; Q8FMR1; Q8TKM8; Q5YX61; Q31D04;Q46YI9; Q98LB3; Q49UX2; Q5NY71; Q7WFQ5; Q89HA2; Q5YRV8; Q9YG88; Q8UE43;Q8YTE6; O67009; O29248; Q81S26; Q9XBI3; Q81F26; Q63CV3; Q5L9I8; Q64PS6;Q9K8E4; Q6HKA0; Q651B0; Q5WEM9; P51785; Q8A608; Q6G543; Q8G3H2; Q7VRL8;Q491Z0; Q57FS2; Q8YEN0; Q8G353; P57656; O51887; P59426; Q9RQ56; Q9RQ48;Q9RQ52; Q62LG7; Q3JV12; Q63WB9; Q9PJ98; Q5HXE4; Q3AER0; P55186; Q3APB9;Q8KER4; Q7NYJ7; Q97EE3; P31959; Q47UN7; Q6NHN6; Q8NQZ9; Q4JUN3; Q47JC0;Q3Z888; Q3ZXH9; Q9RV97; Q317H9; Q725Q1; Q8XAV1; Q8FBR5; P05791; Q6CZC7;Q5NH32; Q5KYA5; Q74BW7; Q7NGK1; Q5FN26; Q4QMF8; P44851; Q5V545; Q7VHW3;Q02139; Q6AEN9; Q72TC0; Q8F219; Q92A32; Q71Y38; Q8Y5S2; Q65QD4; Q46AU2;Q606D6; Q58672; Q8TW40; Q8Q078; Q6M0F3; O27498; P65155; O06069; Q73TT7;P65154; Q31MV2; Q5F8G6; Q9JUE0; Q9JS61; Q82XY7; Q3J9N3; Q3SW60; Q8EN63;P57957; Q3A3A5; Q4FM19; Q7MYJ5; Q6LLH7; Q6KZ30; Q7VC95; Q7TV16; Q7V1T1;Q46LF6; Q48PA6; Q916E0; Q4K498; Q3K559; Q88CQ2; Q87V83; Q4ZZ83; Q4FS54;Q9UZ03; Q8ZYU6; Q8U297; Q8XWR1; Q92M28; Q7UJ69; P31874; Q6N9S5; Q31XP4;Q57HU7; Q5PK00; Q8Z377; P40810; Q8E9D9; Q31UL3; Q329V0; Q83PI6; Q3YVJ3;Q5LN98; Q5HEE8; P65156; P65157; Q6GF19; Q6G7Q4; P65158; Q5HMG3; Q8CNL6;Q4L7T6; Q82E99; O69198; Q8DRT7; P65159; P65160; Q5LYH1; Q5M334; Q4J860;Q97UB2; Q96YK0; Q67KX6; Q8DK13; Q5N3N2; Q7U763; P74689; Q47MS7; Q9WZ21;Q72JA8; Q5SIY0; Q8RDJ9; Q8KTS9; Q83HI6; Q83GP9; Q9 KVW0; Q5E1P2; Q87KB6; Q8DDG1; Q7MGI8; Q7MAN4; Q8PQI0; Q3BYS5; Q4UZT2; Q8PDJ3; Q5GUY8;Q9PH47; Q87F63; Q8ZAB3; Q66G45; and Q5NLJ4.

The conversion of 2-methyl-butyryl-CoA to 2-keto-3-methylvalerate iscatalyzed by branched-chain α-ketoacid dehydrogenase complex (EC1.2.4.4; EC 2.3.1.268; EC 1.8.1.4), which may be encoded by, but notlimited to, one or more of the following genes: P37940; P11178; P12694;Q8HXY4; P50136; A5A6H9; Q9I1M2; P09060; P11960; Q72GU1; Q5SLR4; P37941;P21839; P21953; Q9I1M1; P09061; P35738; Q72GU2; Q5SLR3; P37942; P11181;P11182; P53395; Q911M0; P09062; Q9M5K3; P11959; P21880; Q9I1L9; P09063;Q9M5K2; P54533; Q5UYG6; Q913D1; P31052; O34324; Q5UWH2; Q9HUY1; P31046;P35484; P18925; P57303; Q8K9T7; Q89AQ8; P49819; Q9PJI3; Q9Z773; Q8KCW2;O84561; O50311; Q8CIZ7; P0A9P2; P0A9P1; P0A9P0; P43784; Q9HN74; Q04829;P09622; P80647; Q60HG3; O18480; O08749; P66005; P47513; Q50068; P75393;P66004; P31023; P09623; Q5R4B1; P84545; P14218; P52992; Q6P6R2; O05940;P95596; O00087; P0A9P3; P80503; Q5HGY8; P0A0E6; P99084; Q6 GHY9; Q6GAB8;P0A0E8; P0A0E7; P72740; Q04933; P90597; Q9 KPF6; O50286; P09624; andP50970.

The conversion of 2-keto-3-methylvalerate to 2-methyl-1-butanal iscatalyzed by 2-oxo-acid decarboxylase (EC 4.1.1.72) or2-keto-3-methylvalerate decarboxylase (EC 4.1.1.1), which may be encodedby, but not limited to, one or more of the following genes: P83779;Q6FJA3; Q12629; P33149; P28516; A2Y5L9; Q0DHF6; P51850; Q09737; P51845;P06169; Q05326; A2XFI3; Q10MW3; P51851; Q92345; P51846; Q05327; A2YQ76;Q0D3D2; Q9P7P6; P16467; P26263; Q4WXX9; Q2UKV4; P51844; Q0CNV1; P87208;P34734; P33287; and P06672.

The conversion of 2-methyl-1-butanal to 2-methyl-1-butanol is catalyzedby alcohol dehydrogenase (EC 1.1.1.1), which may be encoded by, but notlimited to, one or more of the following genes: P07327; P28469; Q5RBP7;P25405; P00325; Q5R1W2; P14139; P25406; P00327; P00326; O97959; P00328;P80222; P30350; P49645; P06525; P41747; P12311; Q17334; P43067; P48814;Q70UN9; P23991; P19631; P23236; P48586; P09370; P22246; P07161; P12854;P08843; P26325; Q9Z2M2; Q64413; Q64415; P05336; P20369; Q07288; P00333;P00329; P80512; Q9P6C8; Q75ZX4; Q2R8Z5; P12886; P14219; P41680; P25141;O00097; Q03505; P22797; P06757; P14673; P80338; P13603; P00330; Q07264;P20368; P42327; O45687; O94038; P48815; Q70UP5; Q70UP6; P27581; P25720;P23237; P48587; P09369; P07160; P24267; P37686; P54202; Q24803; P10847;P49383; Q9P4C2; P04707; Q4R1E8; Q0ITW7; O13309; P28032; P14674; P00331;P06758; P42328; P25437; P07754; P44557; P10848; P49384; P39450; P14675;P73138; P07246; P08319; P49385; Q9QYY9; Q64563; Q09669; P80468; P10127;Q6XQ67; P38113; P28332; P41681; Q5R7Z8; Q5X195; P40394; Q64437; P41682;O31186; Q7U1B9; P71818; P33744; P0A9Q8; P0A9Q7; P81600; P72324; Q9SK86;Q9SK87; A1L4Y2; Q8VZ49; Q0V7W6; Q8LEB2; Q9FH04; P81601; P39451; O46649;O46650; Q96533; Q3ZC42; Q17335; P46415; P19854; P11766; P93629; P28474;P80360; P81431; A2XAZ3; Q0DWH1; P80572; O19053; P12711; P79896; P80467;Q9NAR7; Q00669; P21518; P25139; P48584; Q00670; P22245; Q9NG42; P28483;P48585; P51551; Q09009; P51549; P21898; Q07588; Q9NG40; Q27404; P10807;P07162; Q09010; P00334; Q00671; P25721; Q00672; P07159; P84328; P37473;P23361; P23277; Q6LCE4; Q9U8S9; Q9GN94; Q24641; P23278; Q03384; P28484;P51550; Q05114; P26719; P17648; P48977; P81786; P14940; P25988; P00332;Q2FJ31; Q2G0G1; Q2YSX0; Q5HI63; Q99W07; Q7A742; Q6GJ63; Q6GBM4; Q8NXU1;Q5HRD6; Q8CQ56; Q4J781; P39462; P50381; Q96XE0; P51552; P32771; P71017;and P33010.

FIG. 4 shows a proposed pathway for the generation of 3-MBO withisovaleryl CoA, leucine, and 2-isopropyl-3-oxosuccinic acid as thestarting material. Each step of the enzymatic pathway is provided with aletter designation which corresponds to an enzyme.

-   -   Step a) corresponds to the conversion of isovaleryl CoA to        2-ketoisocaproic acid,    -   Step b) corresponds to the conversion of leucine to        2-ketoisocaproic acid,    -   Step c) corresponds to the conversion of        2-isopropyl-3-oxosuccinic acid to 2-ketoisocaproic acid,    -   Step d) corresponds to the conversion of 2-ketoisocaproic acid        to 3-methyl-1-butanal, and    -   Step e) corresponds to the conversion of 3-methyl-1-butanal to        3-methyl-1-butanol.

The conversion of isovaleryl-CoA to 2-ketoisocaproate is catalyzed bythe ketoisovalerate dehydrogenase complex (EC 1.2.4.4; EC 2.3.1.268; EC1.8.1.4), which may be encoded by, but not limited to, one or more ofthe following genes: P37940; P11178; P12694; Q8HXY4; P50136; A5A6H9;Q9I1M2; P09060; P11960; Q72GU1; Q5SLR4; P37941; P21839; P21953; Q9I1M1;P09061; P35738; Q72GU2; Q5SLR3; P37942; P11181; P11182; P53395; Q9I1M0;P09062; Q9M5K3; P11959; P21880; Q9I1L9; P09063; Q9M5K2; P54533; Q5UYG6;Q9I3D1; P31052; O34324; Q5UWH2; Q9HUY1; P31046; P35484; P18925; P57303;Q8K9T7; Q89AQ8; P49819; Q9PJI3; Q9Z773; Q8KCW2; O84561; O50311; Q8CIZ7;P0A9P2; P0A9P1; P0A9P0; P43784; Q9HN74; Q04829; P09622; P80647; Q60HG3;O18480; O08749; P66005; P47513; Q50068; P75393; P66004; P31023; P09623;Q5R4B1; P84545; P14218; P52992; Q6P6R2; O05940; P95596; O00087; P0A9P3;P80503; Q5HGY8; P0A0E6; P99084; Q6 GHY9; Q6GAB8; P0A0E8; P0A0E7; P72740;Q04933; P90597; Q9 KPF6; O50286; P09624; and P50970.

The conversion of L-leucine to 2-ketoisocaproate is catalyzed by thebranched-chain amino acid transaminase (EC 2.6.1.42), which may beencoded by, but not limited to, one or more of the following genes:O14370; P38891; P47176; Q93Y32; P54687; P24288; P54690; Q9GKM4; Q9M439;Q5EA40; O15382; O35855; O19098; Q5REP0; O35854; Q9M401; Q9FYA6; Q9LPM9;P54688; O67733; O29329; P39576; P0AB82; P0AB81; P0AB80; P54689; Q9ZJF1;O26004; Q58414; O27481; O32954; Q10399; O86428; Q1RIJ2; Q92126; Q4ULR3;O05970; Q9AKE5; P0A1A6; P0A1A5; Q5HIC1; P63512; P99138; Q6GJB4; Q6GBT3;P63513; Q5HRJ8; Q8CQ78; O86505; P54691; P74921; Q9Y885; and O31461.

The conversion of L-leucine to 2-ketoisocaproate is catalyzed by leucineaminotransferase (EC 2.6.1.6) or leucine dehydrogenase (EC 1.4.1.9),which may be encoded by, but not limited to, one or more of thefollowing genes: P0A393; P0A392; Q53560; P13154; P54531; Q60030.

The conversion of 2-isopropyl-3-oxosuccinate to 2-ketoisocaproate mayoccur spontaneously.

The conversion of 2-ketoisocaproate to 3-methyl-1-butanal is catalyzedby 2-ketoisocaproate decarboxylase (EC 4.1.1.1), which may be encodedby, but not limited to, one or more of the genes discussed above.

The conversion of 3-methyl-1-butanal to 3-methyl-1-butanol is catalyzedby 3-methyl-1-butanal reductase (EC 1.1.1.265) or alcohol dehydrogenase(EC 1.1.1.1), which may be encoded by, but not limited to, one or moreof the genes discussed above.

The recombinant microorganisms disclosed are engineered to contain aplurality of the enzymes illustrated in FIGS. 1 to 4 and discussedabove, with the goal of producing a particular compound or compounds ofinterest. The entire pathway may be introduced exogenously to a hostcell or select portions of the pathway may be introduced to complementexisting enzymatic systems in the host cell. One of skill in the artcould readily engineer polypeptides providing similar enzymatic functionfor any particular step of the pathways. For example, in addition to thesequences provided herein, one could provide comparable enzymaticactivity with a homolog of any of the polypeptides disclosed sharing atleast about 50%, 55%, 60% or 65% amino acid sequence identity, orpreferably at least about 70%, 75%, 80%, 85%, 90% or 95% amino acidsequence identity.

The genes encoding these enzymes are introduced to the host cell usingstandard molecular biology techniques, such as standard expressionvectors. One or more vectors may be used, where one or more of the genesencoding enzymes of the pathway are present.

FIG. 5 shows an overview of some exemplary pathways that can beexploited to produce compounds of interest.

DEFINITIONS

As used herein, the terms “alkyl,” “alkenyl” and “alkynyl” includestraight-chain, branched-chain and cyclic monovalent hydrocarbylradicals, and combinations of these, which contain only C and H whenthey are unsubstituted. Examples include methyl, ethyl, isobutyl,cyclohexyl, cyclopentylethyl, 2-propenyl, 3-butynyl, and the like. Thetotal number of carbon atoms in each such group is sometimes describedherein, e.g., when the group can contain up to ten carbon atoms it canbe represented as C1-10 or as C1-C10 or C1-10. In certain embodiments,alkyl contains 1-10, 1-8, 1-6, 1-4, or 1-2 carbons.

As used herein, “hydrocarbyl residue” refers to a residue which containsonly carbon and hydrogen. The residue may be aliphatic or aromatic,straight-chain, cyclic, branched, saturated or unsaturated, or anycombination of these. The hydrocarbyl residue, when so stated however,may contain heteroatoms in addition to or instead of the carbon andhydrogen members of the hydrocarbyl group itself.

As used herein, “acyl” encompasses groups comprising an alkyl, alkenyl,alkynyl, aryl or arylalkyl radical attached at one of the two availablevalence positions of a carbonyl carbon atom.

“Aromatic” moiety or “aryl” moiety refers to a monocyclic or fusedbicyclic moiety having the well-known characteristics of aromaticity;examples include phenyl and naphthyl. Similarly, “arylalkyl” refers toan aromatic ring system which is bonded to their attachment pointthrough a linking group such as an alkylene. In certain embodiments,aryl is a 5-6 membered aromatic ring, optionally containing one or moreheteroatoms selected from the group consisting of N, O, and S.

“Alkylene” as used herein refers to a divalent hydrocarbyl group;because it is divalent, it can link two other groups together. Typicallyit refers to —(CH₂)_(n)— where n is 1-10, 1-8, 1-6, 1-4, or 1-2. Theopen valences need not be at opposite ends of a chain. Thus —CH(Me)- and—C(Me)₂- may also be referred to as alkylenes, as can a cyclic groupsuch as cyclopropan-1,1-diyl.

“Arylalkyl” groups as used herein are hydrocarbyl groups if they areunsubstituted, and are described by the total number of carbon atoms inthe ring and alkylene or similar linker. Thus a benzyl group is aC7-arylalkyl group, and phenylethyl is a C8-arylalkyl.

“Arylalkyl” refers to an aromatic ring system bonded to their attachmentpoint through a linking group such as an alkylene, including substitutedor unsubstituted, saturated or unsaturated, cyclic or acyclic linkers.Typically the linker is C1-C8 alkylene or a hetero form thereof. Theselinkers may also include a carbonyl group, thus making them able toprovide substituents as an acyl or heteroacyl moiety. An aryl orheteroaryl ring in an arylalkyl or heteroarylalkyl group may besubstituted with the same substituents described above for aryl groups.

Where an arylalkyl or heteroarylalkyl group is described as optionallysubstituted, the substituents may be on either the alkyl or heteroalkylportion or on the aryl or heteroaryl portion of the group. Thesubstituents optionally present on the alkyl or heteroalkyl portion arethe same as those described above for alkyl groups generally; thesubstituents optionally present on the aryl or heteroaryl portion arethe same as those described above for aryl groups generally.

“Optionally substituted” as used herein indicates that the particulargroup or groups being described may have no non-hydrogen substituents,or the group or groups may have one or more non-hydrogen substituents.If not otherwise specified, the total number of such substituents thatmay be present is equal to the number of H atoms present on theunsubstituted form of the group being described. Where an optionalsubstituent is attached via a double bond, such as a carbonyl oxygen(═O), the group takes up two available valences, so the total number ofsubstituents that may be included is reduced according to the number ofavailable valences.

In certain embodiments, optional substituents are selected from thegroup consisting of halo, ═O, OR, NR₂, NO₂, and CN; wherein each R isindependently H, C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl.

“Halo”, as used herein, includes fluoro, chloro, bromo and iodo. Incertain embodiments, halo is fluoro or chloro.

“Attenuate” as used herein means to reduce expression levels of a geneproduct. For example, functional deletion of the gene encoding an enzymecan be used to attenuate an enzyme. A functional deletion is typically amutation, partial or complete deletion, insertion, or other variationmade to a gene sequence or a sequence controlling the transcription of agene sequence, which reduces or inhibits production of the gene product,or renders the gene product non-functional. In some instances afunctional deletion is described as a knock out mutation.

One of ordinary skill in the art will appreciate that there are manymethods of attenuating enzyme activity. For example, attenuation can beaccomplished by introducing amino acid sequence changes via altering thenucleic acid sequence, placing the gene under the control of a lessactive promoter, expressing interfering RNA, ribozymes or antisensesequences that targeting the gene of interest, or through any othertechnique known in the art.

“Carbon source” as used herein generally refers to a substrate orcompound suitable to be used as a source of carbon for prokaryotic orsimple eukaryotic cell growth. Carbon sources can be in various forms,including, but not limited to carboxylic acids (such as succinic acid,lactic acid, acetic acid), alcohols (e.g., ethanol), sugar alcohols(e.g., glycerol), aldehydes, amino acids, carbohydrates, saturated orunsaturated fatty acids, ketones, peptides, proteins, and mixturesthereof. Examples of carbohydrates include monosaccharides (such asglucose, galactose, xylose, arabinose, and fructose), disaccharides(such as sucrose and lactose), oligosaccharides, and polysaccharides(e.g., starch). Polysaccharides such as starch or cellulose or mixturesthereof and unpurified mixtures from renewable feedstocks such as cheesewhey permeate, cornsteep liquor, sugar beet molasses, and barley malt.Additionally the carbon substrate may also be one-carbon substrates suchas carbon dioxide, or methanol for which metabolic conversion into keybiochemical intermediates has been demonstrated. In addition to one andtwo carbon substrates methylotrophic organisms are also known to utilizea number of other carbon containing compounds such as methylamine,glucosamine and a variety of amino acids for metabolic activity. Forexample, methylotrophic yeast are known to utilize the carbon frommethylamine to form trehalose or glycerol (Bellion et al., Microb.Growth C1-Compd., [Int. Symp.], 7th (1993), 415-32. Editor(s): Murrell,J. Collin; Kelly, Don P. Publisher: Intercept, Andover, UK). Similarly,various species of Candida will metabolize alanine or oleic acid (Sulteret al., Arch. Microbiol. 153:485-489 (1990)). Hence it is contemplatedthat the source of carbon utilized in the present invention mayencompass a wide variety of carbon containing substrates and will onlybe limited by the choice of organism. Lignocellulosic material iscontemplated as a suitable carbon source, i.e., plant biomass that iscomposed of cellulose, hemicellulose, and lignin. Biomass may include(1) wood residues (including sawmill and paper mill discards), (2)municipal paper waste, (3) agricultural residues (including corn stoverand sugarcane bagasse), and (4) dedicated energy crops (which are mostlycomposed of fast growing tall, woody grasses). Carbon dioxide (CO₂), andcoal are also contemplated as suitable carbon sources.

“Culture medium” as used herein includes any medium which supportsmicroorganism life (i.e., a microorganism that is actively metabolizingcarbon). A culture medium usually contains a carbon source. The carbonsource can be anything that can be utilized, with or without additionalenzymes, by the microorganism for energy.

“Deletion” as used herein refers to the removal of one or morenucleotides from a nucleic acid molecule or one or more amino acids froma protein, the regions on either side being joined together.

“Detectable” as used herein refers to be capable of having an existenceor presence ascertained.

“Methylbutanol” refers to a compound of the formula C₅H₁₂O, and the termincludes stereoisomers thereof. Non-limiting examples of structuralisomers of 2-methyl-1-butanol and 3-methyl-1-butanol.

“Endogenous” as used herein in reference to a nucleic acid molecule anda particular cell or microorganism refers to a nucleic acid sequence orpeptide that is in the cell and was not introduced into the cell usingrecombinant engineering techniques. For example, a gene that was presentin the cell when the cell was originally isolated from nature. A gene isstill considered endogenous if the control sequences, such as a promoteror enhancer sequences that activate transcription or translation havebeen altered through recombinant techniques.

“Exogenous” as used herein with reference to a nucleic acid molecule anda particular cell refers to any nucleic acid molecule that does notoriginate from that particular cell as found in nature. Thus, anon-naturally-occurring nucleic acid molecule is considered to beexogenous to a cell once introduced into the cell. A nucleic acidmolecule that is naturally-occurring also can be exogenous to aparticular cell. For example, an entire coding sequence isolated fromcell X is an exogenous nucleic acid with respect to cell Y once thatcoding sequence is introduced into cell Y, even if X and Y are the samecell type.

“Expression” as used herein refers to the process by which a gene'scoded information is converted into the structures and functions of acell, such as a protein, transfer RNA, or ribosomal RNA. Expressed genesinclude those that are transcribed into mRNA and then translated intoprotein and those that are transcribed into RNA but not translated intoprotein (for example, transfer and ribosomal RNAs).

“Hydrocarbon” as used herein includes chemical compounds that containingthe elements carbon (C) and hydrogen (H). Hydrocarbons consist of acarbon backbone and atoms of hydrogen attached to that backbone.Sometimes, the term is used as a shortened form of the term “aliphatichydrocarbon.” There are essentially three types of hydrocarbons: (1)aromatic hydrocarbons, which have at least one aromatic ring; (2)saturated hydrocarbons, also known as alkanes, which lack double, tripleor aromatic bonds; and (3) unsaturated hydrocarbons, which have one ormore double or triple bonds between carbon atoms. Alkenes are chemicalcompounds containing at least one double bond between carbon atoms andalkynes are chemical compounds containing at least one triple bondbetween carbon atoms.

“Isolated” as in “isolated” biological component (such as a nucleic acidmolecule, protein, or cell) refers to the component that has beensubstantially separated or purified away from other biologicalcomponents in which the component naturally occurs, such as otherchromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acidmolecules and proteins that have been “isolated” include nucleic acidmolecules and proteins purified by standard purification methods. Theterm also embraces nucleic acid molecules and proteins prepared byrecombinant expression in a host cell as well as chemically synthesizednucleic acid molecules and proteins.

“Microorganism” as used herein includes prokaryotic and eukaryoticmicrobial species. The terms “microbial cells” and “microbes” are usedinterchangeably with the term microorganism.

“Nucleic Acid Molecule” as used herein encompasses both RNA and DNAmolecules including, without limitation, cDNA, genomic DNA and mRNA.Includes synthetic nucleic acid molecules, such as those that arechemically synthesized or recombinantly produced. The nucleic acidmolecule can be double-stranded or single-stranded. Wheresingle-stranded, the nucleic acid molecule can be the sense strand orthe antisense strand. In addition, nucleic acid molecule can be circularor linear.

“Over-expressed” as used herein refers to when a gene is caused to betranscribed at an elevated rate compared to the endogenous transcriptionrate for that gene. In some examples, over-expression additionallyincludes an elevated rate of translation of the gene compared to theendogenous translation rate for that gene. Methods of testing forover-expression are well known in the art, for example transcribed RNAlevels can be assessed using rtPCR and protein levels can be assessedusing SDS page gel analysis.

“Purified” as used herein does not require absolute purity; rather, itis intended as a relative term.

“Recombinant” as used herein in reference to a recombinant nucleic acidmolecule or protein is one that has a sequence that is not naturallyoccurring, has a sequence that is made by an artificial combination oftwo otherwise separated segments of sequence, or both. This artificialcombination can be achieved, for example, by chemical synthesis or bythe artificial manipulation of isolated segments of nucleic acidmolecules or proteins, such as genetic engineering techniques.Recombinant is also used to describe nucleic acid molecules that havebeen artificially manipulated, but contain the same regulatory sequencesand coding regions that are found in the organism from which the nucleicacid was isolated. A recombinant cell or microorganism is one thatcontains an exogenous nucleic acid molecule, such as a recombinantnucleic acid molecule.

“Spent medium” or “spent culture medium” as used herein refers toculture medium that has been used to support the growth of amicroorganism.

“Stereoisomers” as used herein are isomeric molecules that have the samemolecular formula and connectivity of bonded atoms, but which differ inthe three dimensional orientations of their atoms in space. Non-limitingexamples of stereoisomers are enantiomers, diastereomers, cis-transisomers and conformers.

“Transformed or recombinant cell” as used herein refers to a cell intowhich a nucleic acid molecule has been introduced, such as an acyl-CoAsynthase encoding nucleic acid molecule, for example by molecularbiology techniques. Transformation encompasses all techniques by which anucleic acid molecule can be introduced into such a cell, including, butnot limited to, transfection with viral vectors, conjugation,transformation with plasmid vectors, and introduction of naked DNA byelectroporation, lipofection, and particle gun acceleration.

“Fermentation conditions” referred to herein usually include temperatureranges, levels of aeration, and media selection, which when combinedallow the microorganism to grow. Exemplary media include broths or gels.Generally, the medium includes a carbon source such as glucose,fructose, cellulose, or the like that can be metabolized by themicroorganism directly, or enzymes can be used in the medium tofacilitate metabolizing the carbon source. To determine if cultureconditions permit product production, the microorganism can be culturedfor 24, 36, or 48 hours and a sample can be obtained and analyzed. Forexample, the cells in the sample or the medium in which the cells weregrown can be tested for the presence of the desired product.

“Vector” as used herein refers to a nucleic acid molecule as introducedinto a cell, thereby producing a transformed cell. A vector can includenucleic acid sequences that permit it to replicate in the cell, such asan origin of replication. A vector can also include one or moreselectable marker genes and other genetic elements known in the art.

“Finished fuel” is defined as a chemical compound or a mix of chemicalcompounds (produced through chemical, thermochemical or biologicalroutes) that is in an adequate chemical and physical state to be useddirectly as a neat fuel or fuel additive in an engine. In many cases,but not always, the suitability of a finished fuel for use in an engineapplication is determined by a specification which describes thenecessary physical and chemical properties that need to be met. Someexamples of engines are: internal combustion engine, gas turbine, steamturbine, external combustion engine, and steam boiler. Some examples offinished fuels include: diesel fuel to be used in a compression-ignited(diesel) internal combustion engine, jet fuel to be used in an aviationturbine, fuel oil to be used in a boiler to generate steam or in anexternal combustion engine, ethanol to be used in a flex-fuel engine.Examples of fuel specifications are ASTM standards, mainly used ion theUS, and the EN standards, mainly used in Europe.

“Fuel additive” refers to a compound or composition that is used incombination with another fuel for a variety of reasons, which includebut are not limited to complying with mandates on the use of biofuels,reducing the consumption of fossil fuel-derived products or enhancingthe performance of a fuel or engine. For example, fuel additives can beused to alter the freezing/gelling point, cloud point, lubricity,viscosity, oxidative stability, ignition quality, octane level, andflash point. Additives can further function as antioxidants,demulsifiers, oxygenates, thermal stability improvers, cetane improvers,stabilizers, cold flow improvers, combustion improvers, anti-foams,anti-haze additives, icing inhibitors, injector cleanliness additives,smoke suppressants, drag reducing additives, metal deactivators,dispersants, detergents, demulsifiers, dyes, markers, staticdissipaters, biocides, and/or corrosion inhibitors. One of ordinaryskill in the art will appreciate that MBO and MBO derivatives describedherein can be mixed with one or more fuel or such fuel additives toreduce the dependence on fossil fuel-derived products and/or to impart adesired quality and specific additives are well known in the art. Inaddition, MBO and MBO derivatives can be used themselves as additives inblends with other fuels to impart a desired quality.

Non-limiting examples of additives to the fuel composition of theinvention include: Hybrid compound blends such as combustion catalyst(organo-metallic compound which lowers the ignition point of fuel in thecombustion chamber reducing the temperature burn from 1200 degrees to800° F.), Burn rate modifier (increases the fuel burn time result in anapprox. 30% increase of the available BTUs from the fuel), ethanol as anoctane enhancer to reduce engine knock, biodiesel, polymerization(increases fuel ignition surface area resulting in increased power fromignition), Stabilizer/Demulsifier (prolongs life of fuel and preventswater vapor contamination), Corrosion inhibitor (prevents tankcorrosion), Detergent agent (clean both gasoline and diesel engines withreduced pollution emissions), Catalyst additive (prolongs engine lifeand increases fuel economy), and Detergent (cleans engine); oxygenates,such as methanol, ethanol, isopropyl alcohol, n-butanol, gasoline gradet-butanol, methyl t-butyl ether, tertiary amyl methyl ether, tertiaryhexyl methyl ether, ethyl tertiary butyl ether, tertiary amyl ethylether, and diisopropyl ether; antioxidants, such as, Butylatedhydroxytoluene (BHT), 2,4-Dimethyl-6-tert-butylphenol,2,6-Di-tert-butylphenol (2,6-DTBP), Phenylene diamine, and Ethylenediamine; antiknock agents, such as, Tetra-ethyl lead,Methylcyclopentadienyl manganese tricarbonyl (MMT), Ferrocene, and Ironpentacarbonyl, Toluene, isooctane; Lead scavengers (for leadedgasoline), such as, Tricresyl phosphate (TCP) (also an AW additive andEP additive), 1,2-Dibromoethane, and 1,2-Dichloroethane; and Fuel dyes,such as, Solvent Red 24, Solvent Red 26, Solvent Yellow 124, and SolventBlue 35. Other additives include, Nitromethane (increases engine power,“nitro”), Acetone (vaporization additive, mainly used with methanolracing fuel to improve vaporisation at start up), Butyl rubber (aspolyisobutylene succinimide, detergent to prevent fouling of diesel fuelinjectors), Ferox (catalyst additive that increases fuel economy, cleansengine, lowers emission of pollutants, prolongs engine life), Ferrouspicrate (improves combustion, increases mileage), Silicones(anti-foaming agents for diesel, damage oxygen sensors in gasolineengines), and Tetranitromethane (to increase cetane number of dieselfuel).

In certain embodiments, the invention provides for a fuel compositioncomprising MBO or a derivative thereof as described herein and one ormore additives. In certain embodiments, the additives are at least 1-5%,1-10%, 1-15%, 1-20%, 1-25%, 1-30%, 1-35%, 1-40%, 1-45%, 1-50%, 1-55%,1-60%, 1-65%, 1-70%, 1-75%, 1-80%, 1-85%, 1-90%, 1-95%, or 1-100% of theweight of the composition. In certain embodiments, the additivescomprise 1-5%, 1-10%, 1-15%, 1-20%, 1-25%, 1-30%, 1-35%, 1-40%, 1-45%,1-50%, 1-55%, 1-60%, 1-65%, 1-70%, 1-75%, 1-80%, 1-85%, 1-90%, 1-95%, or1-100% of the volume of the composition. In certain embodiments, theadditives comprise 5-10%, 10-30%, or 25-40% of the weight of thecomposition. In certain embodiments, the additives comprise 5-10%,10-30%, or 25-40% of the volume of the composition.

In certain embodiments, the additives are at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%,21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%,49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the weight of thecomposition.

In certain embodiments, the additives are at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%,21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%,49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the volume of thecomposition.

One of ordinary skill in the art will also appreciate that the MBO andMBO derivatives described herein are can be mixed with other fuels suchas bio-diesel, various alcohols such as ethanol and butanol, andpetroleum-based products such as gasoline. In certain embodiments, theconventional petroleum-based fuel is at least 10%, 20%, 30%, 40%, 50%,60%, 75%, 85%, 95%, or 99% of the weight or volume of the composition.

In one embodiment, the compounds of the present invention andderivatives thereof can themselves provide a fuel composition, whereinthe compound or a combination of compounds of the present inventioncomprise approximately 100% of the fuel composition. In various otherembodiments the compounds of the present invention and derivativesthereof are combined with other fuels or biofuels to provide a fuelcomposition, wherein the compound of a combination of compounds of thepresent invention comprise 1-99% of the weight or 1-99% or the volume ofthe composition, any specific percentage in the given range, or anypercentage sub-range within the given range.

In one embodiment the compounds of the present invention are combinedwith a petroleum-based fuel, for example, gasoline, diesel, jet fuel,kerosene, heating oil or any combinations thereof, to provide a fuelcomposition. In a specific embodiment, MBO is combined with gasoline,with the purpose of providing oxygen and increasing the octane contentof the fuel composition. In another specific embodiment, an MBO ether iscombined with a petroleum-based diesel, e.g., a distillate, with thepurpose of providing oxygen and increasing cetane content.

In another embodiment the compounds of the present invention arecombined with another biofuel, for example, methanol, ethanol, propanol,butanol or any combinations thereof, to provide a fuel composition.

In another embodiment the compounds of the present invention arecombined with a petroleum-based fuel and another biofuel. In a specificembodiment, MBO is combined with ethanol to reduce the Reid vaporpressure (RVP) of an ethanol-gasoline mixture.

Bio-crudes are biologically produced compounds or a mix of differentbiologically produced compounds that are used as a feedstock forpetroleum refineries in replacement of, or in complement to, crude oil.In general, but not necessarily, these feedstocks have beenpre-processed through biological, chemical, mechanical or thermalprocesses in order to be in a liquid state that is adequate forintroduction in a petroleum refinery.

Microbial Hosts

Microbial hosts of the invention may be selected from but not limited toarchaea, bacteria, cyanobacteria, fungi, yeasts, thraustochytrids andphotosynthetic microorganisms. In certain embodiments, examples ofcriteria for selection of suitable microbial hosts include thefollowing: intrinsic tolerance to desired product, high rate of glucoseor alternative carbon substrate utilization, availability of genetictools for gene manipulation, and the ability to generate stablechromosomal alterations. However, the present invention should not beinterpreted to be limited by these criteria.

The microbial host used for MBO or MBO derivative production ispreferably tolerant to MBO or MBO derivatives so that the yield is notlimited by product toxicity. Suitable host strains with a tolerance forMBO or MBO derivatives may be identified by screening based on theintrinsic tolerance of the strain. The intrinsic tolerance of microbesto MBO or MBO derivatives may be measured by determining theconcentration of MBO or MBO derivatives that is responsible for 50%inhibition of the growth rate (IC₅₀) when grown in a minimal culturemedium. The IC₅₀ values may be determined using methods known in theart. For example, the microbes of interest may be grown in the presenceof various amounts of MBO or MBO derivatives and the growth ratemonitored by measuring the optical density. The doubling time may becalculated from the logarithmic part of the growth curve and used as ameasure of the growth rate. The concentration of MBO or of the MBOderivative that produces 50% inhibition of growth may be determined froma graph of the percent inhibition of growth versus the concentration ofMBO or MBO derivative. In some embodiments, the host strain should havean IC₅₀ for MBO or MBO derivative of greater than 0.5%. The IC₅₀ valuecan be similarly calculated for microbes in contact with compounds otherthan MBO.

The ability to genetically modify the host is essential for theproduction of any recombinant microorganism. The mode of gene transfertechnology may be by electroporation, conjugation, transduction ornatural transformation. A broad range of host conjugative plasmids anddrug resistance and nutritional markers are available. The cloningvectors are tailored to the host organisms based on the nature ofantibiotic resistance markers that can function in that host.

In some embodiments, the microbial host also may be manipulated in orderto inactivate competing pathways for carbon flow by deleting variousgenes. This may require the ability to direct chromosomal integrationevents. Additionally, the production host should be amenable to chemicalmutagenesis so that mutations to improve intrinsic product tolerance maybe obtained.

Microbial hosts of the invention may be selected from but not limited toarchaea, bacteria, cyanobacteria, fungi, yeasts, thraustochytrids andphotosynthetic microorganisms. Examples of suitable microbial hosts foruse with the disclosed invention include, but are not limited to,members of the genera Clostridium, Zymomonas, Escherichia, Salmonella,Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus,Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium,Brevibacterium, Pichia, Candida, Hansenula, and Saccharomyces. Examplesof particular bacteria hosts include but are not limited to Escherichiacoli, Corynebacterium glutamicum, Pseudomonas putida, Bacillus subtilis,Rhodopseudomonas palustris, Rhodobacter sphaeroides, Micrococcus luteus,Streptomyces coelicolor, Streptomyces griseus, Lactobacillus fermentum,Lactococcus lactis, Lactobacillus bulgaricus, Acetobacter xylinum,Streptococcus lactis, Bacillus stearothermophilus, Propionibactershermanii, Streptococcus thermophilus, Deinococcus radiodurans, Delftiaacidovorans, Enterococcus faecium, Pseudomonas mendocina, and Serratiamarcescens. Examples of particular yeast hosts include but are notlimited to Saccharomyces cerevisiae, Saccharomyces carlsbergensis,Kluyveromyces lactis, Kluyveromyces marxianus, Yarrowia lipolytica,Debaryomyces hansenii, Ashbya gossypii, ZygoSaccharomyces rouxii,ZygoSaccharomyces bailii, Brettanomyces bruxellensis,SchizoSaccharomyces pombe, Rhodotorula glutinis, Pichia stipitis, Pichiapastoris, Candida tropicalis, Candida utilis and Candida guilliermondii.Examples of particular fungal hosts include but are not limited toAspergillus niger, Aspergillus oryzae, Neurospora crassa, Fusariumvenenatum and Penicillium chrysogenum. Examples of particularphotosynthetic microorganism hosts include but are not limited toAnabaena sp., Chlamydomonas reinhardtii, Chlorella sp., Cyclotella sp.,Gloeobacter violaceus, Nannochloropsis sp., Nodularia sp., Nostoc sp.,Prochlorococcus sp., Synechococcus sp., Oscillatoria sp., Arthrospirasp., Lyngbya sp., Dunaliella sp., and Synechocystis sp. Examples ofparticular thraustochytrid hosts include but are not limited toSchizochytrium sp. and Thraustochytrium sp.

Construction of Production Host

Recombinant organisms containing the necessary genes that will encodethe enzymatic pathway for the conversion of a carbon source to MBO oranother compound of interest may be constructed using techniques wellknown in the art. In the present invention, genes encoding the enzymesof one of the MBO biosynthetic pathways of the invention may be isolatedfrom various sources. Non-limiting examples of enzymes which can be usedare discussed above.

Methods of obtaining desired genes from a bacterial genome are commonand well known in the art of molecular biology. For example, if thesequence of the gene is known, suitable genomic libraries may be createdby restriction endonuclease digestion and may be screened with probescomplementary to the desired gene sequence. Once the sequence isisolated, the DNA may be amplified using standard primer-directedamplification methods such as polymerase chain reaction (U.S. Pat. No.4,683,202) to obtain amounts of DNA suitable for transformation usingappropriate vectors. Tools for codon optimization for expression in aheterologous host are readily available. Some tools for codonoptimization are available based on the GC content of the host organism.

Once the relevant pathway genes are identified and isolated they may betransformed into suitable expression hosts by means well known in theart. Vectors or cassettes useful for the transformation of a variety ofhost cells are common and commercially available from companies such asEPICENTRE® (Madison, Wis.), Invitrogen Corp. (Carlsbad, Calif.),Stratagene (La Jolla, Calif.), and New England Biolabs, Inc. (Beverly,Mass.). Typically the vector or cassette contains sequences directingtranscription and translation of the relevant gene, a selectable marker,and sequences allowing autonomous replication or chromosomalintegration. Suitable vectors comprise a region 5′ of the gene whichharbors transcriptional initiation controls and a region 3′ of the DNAfragment which controls transcriptional termination. Both controlregions may be derived from genes homologous to the transformed hostcell, although it is to be understood that such control regions may alsobe derived from genes that are not native to the specific species chosenas a production host.

Initiation control regions or promoters, which are useful to driveexpression of the relevant pathway coding regions in the desired hostcell are numerous and familiar to those skilled in the art. Virtuallyany promoter capable of driving these genetic elements is suitable forthe present invention including, but not limited to, TEF, CYC1, HIS3,GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI,CUP1, FBA, GPD, and GPM (useful for expression in Saccharomyces); AOX1(useful for expression in Pichia); and lac, ara, tet, trp, IP_(L),IP_(R), T7, tac, and trc (useful for expression in Escherichia coli,Alcaligenes, and Pseudomonas); the amy, apr, npr promoters and variousphage promoters useful for expression in Bacillus subtilis, Bacilluslicheniformis, and Paenibacillus macerans; nisA (useful for expressionGram-positive bacteria, Eichenbaum et al. Appl. Environ. Microbiol.64(8):2763-2769 (1998)); and the synthetic P11 promoter (useful forexpression in Lactobacillus plantarum, Rud et al., Microbiology152:1011-1019 (2006)).

Termination control regions may also be derived from various genesnative to the preferred hosts. Optionally, a termination site may beunnecessary.

Certain vectors are capable of replicating in a broad range of hostbacteria and can be transferred by conjugation. The complete andannotated sequence of pRK404 and three related vectors-pRK437, pRK442,and pRK442(H) are available. These derivatives have proven to bevaluable tools for genetic manipulation in Gram-negative bacteria (Scottet al., Plasmid 50(1):74-79 (2003)). Several plasmid derivatives ofbroad-host-range Inc P4 plasmid RSF1010 are also available withpromoters that can function in a range of Gram-negative bacteria.Plasmid pAYC36 and pAYC37, have active promoters along with multiplecloning sites to allow for the heterologous gene expression inGram-negative bacteria.

Chromosomal gene replacement tools are also widely available. Forexample, a thermosensitive variant of the broad-host-range repliconpWV101 has been modified to construct a plasmid pVE6002 which can beused to effect gene replacement in a range of Gram-positive bacteria(Maguin et al., J. Bacteriol. 174(17):5633-5638 (1992)). Additionally,in vitro transposomes are available to create random mutations in avariety of genomes from commercial sources such as EPICENTRE®.

Culture Media and Conditions

Culture medium in the present invention contains suitable carbon source.In addition to an appropriate carbon source, culture medium typicallycontains suitable minerals, salts, cofactors, buffers and othercomponents, known to those skilled in the art, suitable for the growthof the cultures and promotion of the enzymatic pathway necessary for MBOproduction, as well as the production of other compounds.

Typically cells are grown at a temperature in the range of 25° C. to 40°C. in an appropriate medium. Suitable growth media in the presentinvention are common commercially prepared media such as Luria Bertani(LB) broth, Sabouraud Dextrose (SD) broth or Yeast medium (YM) broth.Other defined or synthetic growth media may also be used, and theappropriate medium for growth of the particular microorganism will beknown by one skilled in the art of microbiology or fermentation science.Suitable pH ranges for the fermentation are between pH 5.0 to pH 9.0. Insome embodiments the initial pH is 6.0 to pH 8.0. Microorganism culturemay be performed under aerobic, anaerobic, or microaerobic conditions.

Synthesis of Ethers from 2-Methyl-1-Butanol or 3-Methyl-1-Butanol

Oxygenated additives can be used to boost the performance of fuels.Ethers have a much lower water absorbance than alcohols and can be usedas a cetane enhancer. One method of the preparation of ethers is theintermolecular condensation of an alcohol using an acid catalyst. Thismethod is used industrially. U.S. Pat. No. 6,218,583 (Apr. 17, 2001)describes the production of n-pentyl ether.

In one aspect, the invention provides a method to chemically convertbiosynthetically prepared 2-methyl-1-butanol and 3-methyl-1-butanol totheir corresponding ethers, 1-(isopentyloxy)-3-methylbutane and2-methyl-1-(2-methylbutoxy)butane respectively. The structures are shownbelow.

These two ethers are also be referred to as bis-(3-methylbutyl)ether andbis-(2-methylbutyl)ether, respectively. ‘Methylbutanol’ as used herein,refers to either 2

In one embodiment, the conversion step of converting methylbutanol tomethylbutyl ether comprises treating the methylbutanol with an acidresulting in the formation of methylbutyl ether. Non-limiting examplesof acids include hydrochloric acid, hydrobromic acid, sulfuric acid,nitric acid, phosphoric acid, chromic acid, sulfonic acids (methane-,ethane-, benzene-, toluene-, trifluoromethyl-), perfluoroalkane sulfonicacids. In one embodiment, the acid is trifluoromethyl sulfonic acid,CF₃SO₃H. In alternative embodiments, the reaction is a heterogeneousmixture and takes place with a solid phase acid catalyst, polymer-bound,or resin-bound acid catalyst. The advantage of this method is that theacid catalyst is easily separated from the reaction mixture.

In certain embodiments, the catalytic amount of acid at the start of thereaction sequence is between 0.001 and 0.20 molar equivalents relativeto the alcohol. In other embodiments, the catalytic amount of acid isbetween 0.001 and 0.05 molar equivalents relative to the alcohol. Inother embodiments, the catalytic amount of acid is less than 0.04 molarequivalents relative to the alcohol. In other embodiments, the catalyticamount of acid is less than 0.03 molar equivalents relative to thealcohol. In other embodiments the catalytic amount of acid is between0.015 and 0.035 molar equivalents relative to the alcohol.

In certain embodiments, the reaction temperature is between 75-400° C.In certain embodiments the reaction temperature is between 75-150° C. Incertain embodiments the reaction temperature is between the temperatureof the boiling point of the ether and that of the alcohol(2-methyl-1-butanol b.p. 128° C.; 3-methyl-1-butanol b.p. 132° C. Incertain embodiments the reaction temperature is the boiling point of thereaction mixture and thus varies depending on the composition of thereaction mixture.

In one embodiment, the conversion step of converting methylbutanol tomethylbutyl ether comprises refluxing a solution comprisingmethylbutanol and a catalytic amount of acid, and removal of watergenerated from the solution. Removal of the byproduct water generated inthe dehydration reaction can be carried out by distillation and shiftsthe chemical equilibrium in favor of ether formation. Toward the end ofthe reaction, the mixture may be further neutralized and the etherproduct isolated. The product may be isolated through any techniqueknown in the art such as extraction, filtration, chromatography,distillation, vacuum distillation or any combination thereof.

Removal of the water generated during the reaction can be carried outwith a Dean-Stark or Dean-stark-like apparatus. The Dean-Stark apparatusor Dean-Stark receiver or distilling trap is a piece of laboratoryglassware used in synthetic chemistry to collect water (or occasionallyother liquid) from a reactor. It is used in combination with a refluxcondenser and a batch reactor for continuous removal of the water thatis produced during a chemical reaction performed at reflux temperature.

The progress of the reaction may be monitored by sampling the reactionmixture and analyzing the composition. Analysis can be carried out witha number of analytical techniques or instruments such as gaschromatography, high pressure liquid chromatography, nuclear magneticresonance spectroscopy, and mass spectroscopy. An estimate of reactionprogress can also be determined by separating and measuring the amountof water by-product from the reaction mixture.

In one embodiment, the methylbutyl ether product isbis-(3-methylbutyl)ether, bis-(2-methylbutyl)ether,1-(isopentyloxy)-2-methylbutane, or any combination thereof. Reaction ofa single alcohol species produces the corresponding symmetrical ether,such as bis-(3-methylbutyl)ether, bis-(2-methylbutyl)ether. Reaction ofalcohols that are composed of mixtures of alcohols, such as3-methyl-1-butanol and 2-methyl-1-butanol, can result in the formationof both symmetrical and mixed ethers. 1-(isopentyloxy)-2-methylbutane isan example of the mixed ether.

Alternatively, ethers can be produced by Williamson ether synthesis.This synthesis consists of a bimolecular nucleophilic substitutionreaction between a sodium alkoxide with an alkyl halide, alkylsulfonate, or alkyl sulfate. For example, the reaction of1-bromo-3-methyl butane and sodium-2-methylbutan-1-olate yields themixed ether. Likewise, the reaction of sodium-2-methylbutan-1-olate with1-bromo-2-methylbutane yields the symmetrical bis-(2-methylbutyl)etherand the reaction of sodium-3-methylbutan-1-olate with1-bromo-3-methylbutane yields the symmetrical bis-(3-methylbutyl)ether.Reaction conditions can be determined experimentally by a person havingordinary skill in the art.

Carbon Fingerprinting

Compositions that are derived from the biosynthetic methods describedherein can be characterized by carbon fingerprinting, and their lack ofimpurities when compared to petroleum derived fuels. Carbonfingerprinting is valuable in distinguishing MBO and other compounds ofinterest by the biosynthetic methods described herein from othermethods.

Biologically produced compounds of interest described here represent anew source of fuels, such as alcohols, diesel, and gasoline. These newfuels can be distinguished from fuels derived form petrochemical carbonon the basis of dual carbon-isotopic fingerprinting. Additionally, thespecific source of biosourced carbon (e.g., glucose vs. glycerol) can bedetermined by dual carbon-isotopic fingerprinting (see U.S. Pat. No.7,169,588, which is herein incorporated by reference in its entirety, inparticular, see col. 4, line 31, to col. 6, line 8).

The compounds of interest and the associated biofuels, chemicals, andmixtures may be completely distinguished from their petrochemicalderived counterparts on the basis of ¹⁴C (f_(M)) and dualcarbon-isotopic fingerprinting.

The compounds of interest described herein have utility in theproduction of biofuels, chemicals, and biochemicals. For example, MBOand derivatives thereof can be used as a solvent, and in the flavor andfragrance industry. The new products provided by the instant inventionadditionally may be distinguished on the basis of dual carbon-isotopicfingerprinting from those materials derived solely from petrochemicalsources. The ability to distinguish these products is beneficial intracking these materials in commerce. For example, fuels or chemicalscomprising both “new” and “old” carbon isotope profiles may bedistinguished from fuels and chemicals made only of “old” materials.Thus, the instant materials may be followed in commerce or identified incommerce as a biofuel on the basis of their unique profile. In addition,other competing materials can be identified as being biologicallyderived or derived from a petrochemical source.

The compounds of interest described herein have further utility in theproduction of biodiesels, for example, for transesterification ofvegetable oil, animal fats, or wastes thereof. Further uses of thecompounds described herein are readily known to one of skill in the art,for example, general chemical uses, and uses as a solvent.

In a non-limiting example, a biofuel composition is made that includescompounds of interest having δ13C of from about −10.9 to about −15.4,wherein the compound or compounds accounts for at least about 85% ofbiosourced material (i.e., derived from a renewable resource such ascellulosic materials and sugars) in the composition.

The following examples are offered to illustrate but not to limit theinvention.

Example 1 Increase of Intracellular Threonine

As series of genes encoding enzymes from glucose to threonine werecloned from Saccharomyces cerevisiae and Pichia stipitis. These geneswere tested for functional activity, either by enzyme assay orcomplementation of deletion mutations in Saccharomyces cerevisiae andprofiling of intracellular amino acids.

Below are a series of experiments that highlight genes required forelevated threonine production FIGS. 6 through 16 show amino acidproduction by the overexpression of pathway genes in particular deletionbackgrounds.

Amino Acid Analysis of MDH2 (7432), THR1 (7239) and PCK1 (8110)Expressed from p415TEF in Strain 7123

The impact of various expression vectors encoding different genes ofinterest on amino acid levels was made. For this analysis, constructscontaining the MDH2 (7432), THR1 (7239) and PCK1 (8110) genes on thep415TEF expression vector were introduced into host strain 7123 (ATCC200869 (MATα ade2Δ::hisG his3Δ200 leu2Δ0 lys2Δ0 met15Δ0 trp1Δ63 ura3Δ0).Strain 7196 (7123 containing empty p415TEF) was included as a control.Cultures were grown overnight in selective medium. Cells were pelleted,washed with sodium phosphate buffer (50 mM, pH 7.0), and extracted bybead beating in warm (50° C.) 80% ethanol. The data is shown in FIG. 6.As depicted, significant differences in L-threonine content wereobserved in cultures expressing MDH2 and PCK1 only. Amino acid valuesare expressed as a percentage of individual amino acids in the totalamino acid content of the cells.

Amino Acid Analysis of HOM3 (7245) and HOM3-R2 (7242) Expressed fromp416TEF in Strain 7123

HOM3 (7245) and HOM3-R2 (7242) expressed from p416TEF in strain 7123were analyzed. Strain 7123 with an empty expression vector(p416TEF)(7209) was included as a control. Cultures were grown overnightin selective medium and extracted as above. The data is shown in FIG. 7.Significantly lower L-threonine content was observed in culturesexpressing HOM3-R2, whereas no significant difference was observed forwild-type HOM3 and the background strain 7209. Amino acid values areexpressed as a percentage of individual amino acids in the total aminoacid content of the cells.

Amino Acid Analysis of S. cerevisiae HOM3 and HOM3-R2 Expressed fromp416TEF or p416CYC in Strain 7790 (BY4741 ΔHOM3::KanMX)

Host organisms containing expression vectors p416TEF-HOM3 (7718),p416TEF-HOM3-R2 (7819), p416CYC-HOM3 (7809), and p416CYC-HOM3-R2 (7805)were prepared. Cultures were grown overnight in a defined medium lackingthreonine and isoleucine to select for HOM3 expression. Cells were grownand extracted as before. The results are shown in FIG. 8. Significantlyhigher L-threonine content was observed in cultures expressing HOM3-R2from the CYC promoter as compared to the TEF promoter. A significantincrease in L-threonine content was also observed when wild-type HOM3was expressed from TEF as compared to the CYC promoter. The totalL-threonine content of the cells in the CYC expressed HOM3-R2 culturereached 30% (wt/wt). Amino acid values are expressed as a percentage ofindividual amino acids in the total amino acid content of the cells.

Amino Acid Analysis of Pichia stipitis and Saccharomyces cerevisiaeHOM2, HOM6 and THR1 Expressed from the TEF Promoter in their RespectiveDeletion Backgrounds

The host organisms used in these experiments were: FIG. 9: 8113=7578(ΔHOM2) with p416TEF, 8114=7578 with p416TEF-PsHOM2, 7717=7578p416TEF-ScHOM2; FIG. 10: 8111=7582 (ΔHOM6) with p415TEF, 8112=7582p415TEF-PsHOM6, 7716=7582 p415TEF-ScHOM6; FIG. 11: 8115=7583 (ΔTHR1)with p415TEF, 8116=7583 p415TEF-PsTHR1, 7715=7583 p415TEF-ScTHR1.Cultures were grown overnight in a defined medium lacking threonine andisoleucine to select for complementation of chromosomal deletions.Complementation of the last gene in the threonine pathway, THR4, was notpossible due to ΔTHR4 strain growing on media lacking threonine andisoleucine. Cells were grown and extracted as described before. Resultsare shown in FIGS. 13 to 15. No significant differences in any aminoacid were observed when compared to the background BY4741 strain. Aminoacid values are expressed as a percentage of individual amino acids inthe total amino acid content of the cells.

Amino Acid Composition in BY4741 Parental Strain and KanMX Deletions ofL-Threonine Pathway Enzymes in that Background

The host organisms used in these experiments were: 7576=(ΔAAT1),7577=(ΔAAT2), 8177=(ΔMDH1) and 7575=(ΔMDH2). Results are shown in FIG.12. Levels of amino acids were not significantly different for anystrain other than ΔAAT2, which produced no detectable threonine andsignificantly higher levels of lysine, glutamate/glutamine and arginine.Amino acid values are expressed as a percentage of individual aminoacids in the total amino acid content of the cells.

Amino Acid Composition in BY4741 Parental Strain and KanMX Deletions ofL-Threonine Pathway Enzymes in that Background

Amino acid analysis of P. stipitis HOM3 (8037) and HOM3-R2 (8038)expressed from p416TEF in strain 7790 (BY4741 ΔHOM3::KanMX) (FIG. 13).Cells were grown in defined medium lacking threonine and isoleucine forapproximately 24 hrs and extracted as previously stated. Levels ofL-threonine were significantly higher in the HOM3-R2 variant, andapproached 35% of the total amino acid content of the cells. Although nodirect comparison was made, in 7790 the S. cerevisiae HOM3-R2 variantexpressed from CYC promoter achieved 30% threonine content compared to35% for the P. stipitis HOM3-R2 mutant expressed from TEF. The wild-typeHOM3 from P. stipitis was also found to produce more threonine thanwild-type HOM3 from S. cerevisiae (15% vs. 10%) in strain 7790. Aminoacid values are expressed as a percentage of individual amino acids inthe total amino acid content of the cells.

Amino Acid Analysis of HOM3 (7718) and HOM3-R7 (8118) Expressed fromp416TEF in Strain 7790 (BY4741 ΔHOM3::KanMX)

Constructs 7718 and 8118 were expressed in strain 7790 (FIG. 14).Cultures were grown for 48 hours in a defined medium lacking threonineand isoleucine to select for HOM3 complementation. The slow growth ofthe cells expressing the R7 mutation necessitated the extra 24 hours ofgrowth compared to cultures expressing the R2 mutation. Cells were grownand extracted as stated previously. Significantly higher 1-threoninecontent was observed in cultures expressing HOM3-R7 from the TEFpromoter compared to the native HOM3. The total 1-threonine content ofthe cells in the TEF expressed HOM3-R7 culture reached 28% (wt/wt).Amino acid values are expressed as a percentage of individual aminoacids in the total amino acid content of the cells.

Co-Expression Amino Acid Analysis

Amino acid analysis of co-expression of AAT1 (7957), AAT2 (7961), MDH1(7958), MDH2 (7962) and PCK (7959) together with HOM3-R2. Strain 7960contained an empty p415TEF plasmid as a control. (FIG. 15.) All enzymeswere expressed from p415TEF, with the exception of HOM3-R2, which wasexpressed from p416CYC. Constructs were made in strain 7768 (BY4741ILV1::TEF-Ilv1-fbr). Cultures were grown for 24 hrs in a defined mediumlacking threonine and isoleucine and cells were prepared for amino acidanalysis as described previously. Levels of amino acids were similar,and threonine content was significantly lower than that found in strainscontaining a wild-type ILV1, indicating that threonine is limiting in2-MBO production. In cells expressing AAT2 and MDH2, higher levels ofhomoserine were observed, consistent with what was observed when HOM3-R2was expressed by itself. Specifically, the 7% homoserine content foundin the culture expressing MDH2 with HOM3-R2 was the highest seen in anyculture tested, indicating THR1 limitation and potentially higher fluxthrough the Asp/Thr pathway than HOM3-R2 alone. Also of note is thesignificantly higher alanine content when PCK is expressed with HOM3-R2,potentially indicating an increase in pyruvate concentration, leading toincreased alanine. Amino acid values are expressed as a percentage ofindividual amino acids in the total amino acid content of the cells.

Amino Acid Analysis of P. stipitis HOM3 and HOM3-R2Expressed fromp416TEF in Strain 7790

An amino acid analysis of P. stipitis HOM3 and HOM3-R2 expressed fromp416TEF in strain 7790 (HOM3=8039, HOM3-R2=8040) and BY4741 (HOM3=8119,HOM3-R2=8120) was conducted (FIG. 16). Cells were grown in definedmedium lacking threonine and isoleucine for approximately 24 hours andextracted as previously stated. Levels of 1-threonine were as observedbefore in the 7790 strain (35% total amino acid content), and expressionof this enzyme in the background of chromosomally encoded S. cerevisiaeHOM3 was not significantly different. The threonine level for wild-typeP. stipitis HOM3 in 7790 was approximately the same as previouslyobserved (˜15%). Surprisingly, this same enzyme expressed in the BY4741background resulted in a significantly higher threonine level ofapproximately 5%, suggesting a contribution from the native HOM3. Thiscould be interpreted as showing a theoretical maximal level of threonineachieved by HOM3-R2, since no additional threonine was detected byexpression in BY4741. Alternatively, it could indicate a limitation inupstream precursors, specifically oxaloacetate, since aspartate levelsappeared constant in the four different experiments. Additionally,alanine levels showed a significant decrease in the HOM3-R2 strains. Onepossible explanation is increased pyruvate flux into the Asp/Thr pathwayvia OAA, thereby limiting direct transamination of pyruvate to alanine.Amino acid values are expressed as a percentage of individual aminoacids in the total amino acid content of the cells.

Example 2 Identification of Genes Relevant for Methylbutanol Production

The Saccharomyces cerevisiae deletion collection was analyze toascertain which specific loci are relevant to 2-MBO production. Variousstrains designated by their gene deletion using nomenclature consistentwith the Saccharomyces Genome Database project were transformed withp415TEF-ILV1^(FBR) and the control empty vector p415TEF. FIGS. 17 and 18show the production of 2-MBO, while FIG. 19 shows the correspondingproduction of 3-MBO. The table below shows data relating to theproduction of 2-MBO in HOM3^(FBR): TEF ILV1^(FBR) host strain with theaddition of key genes in the pyruvate to threonine pathway.

2-MBO Strain Genotype (μM) 8102 TEF HOM3^(FBR): TEF ILV1^(FBR)p413TEF-PCK(Ps) 427.49 p415TEF-THR1(Ps) p416TEF-MDH2(Ps) 8103 TEFHOM3^(FBR): TEF ILV1^(FBR) p413TEF-PCK(Ps) 405.55 p415TEF-HOM6(Ps)p416TEF-PYC(Ps) 8104 TEF HOM3^(FBR): TEF ILV1^(FBR) p413TEF-PCK(Ps)1441.41 p415TEF-HOM6(Ps) p416TEF-MDH2(Ps) 8105 TEF HOM3^(FBR): TEFILV1^(FBR) 1FBR p413TEF-PCK(Ps) 1122.6 p415TEF-THR1(Ps) p416TEF-PYC(Ps)8050 TEF HOM3^(FBR): TEF ILV1^(FBR) p415TEF 2045.39 8107 TEF HOM3^(FBR):TEF ILV1^(FBR) p413TEF-PYC(Ps) 3339.02 p415TEF-THR1(Ps) p416TEF-HOM2(Ps)8108 TEF HOM3^(FBR): TEF ILV1^(FBR) p413TEF-PYC(Ps) 3508.43p415TEF-THR1(Ps) p416TEF-MDH2(Ps)

Example 3 Identification and Modification of Genes Relevant forMethylbutanol Production

Elevated intracellular pools of threonine accumulate in the cytoplasm ofS. cerevisiae. The deamination of threonine is carried out by twoenzymes; a catabolic threonine dehydratase (CHA1; FIG. 20) and amitochondrial threonine deaminase (ILV1; FIG. 21). Experimental evidencehas shown that overexpression of either of these enzymes results inincrease production of 2-methylbutanol (2-MBO) in the growth medium. Theproduction of 2-MBO can be further elevated by the modification of aminoacid sequence ILV1 that are thought to be involved in the binding ofisoleucine and create a feedback mechanism to shut down the pathway whenintracellular amounts of isoleucine are in excess. The committed stepsto isoleucine production and the conversion of threonine take place inthe mitochondria. The key enzymes in the pathway (threonine deaminase,acetolactate synthase, acetohydroxyacid reductoisomerase anddihydroxyacid dehydratase are expressed in the nucleus and translocateto the mitochondria). To alleviate potential redox issues and to createa cytoplasmic isoleucine pathway a bioinformatics study was carried outto remove the mitochondrial targeting sequence or express prokaryoticcounterparts which have no organelle targeting components (Table 1).

Using the TargetP informatics program, attempts were made to predict themitochondrial targeting sequence of the isoleucine pathwaygenes/proteins and to determine which deletions would leave theseenzymes residing within the cytoplasm. Table 2 below shows the TargetPresults.

TABLE 2 TargetP Mitochondrial Targeting Sequence Prediction ofSaccharomyces cerevisiae Isoleucine Pathway Genes Length Loca- Gene(Enzyme) (aa) mTP SP Other tion RC TPlen Ilv1 (threonine 576 0.620 0.0160.408 M 4 31 deaminase) Ilv2 (acetolactate 687 0.949 0.012 0.124 M 1 37synthase-subunit) Ilv6 (acetolactate 309 0.962 0.028 0.057 M 1 24synthase-subunit) Ilv5 395 0.947 0.012 0.108 M 1 22 (acetohydroxyacidreductoisomerase) Ilv3 dihydroxyacid 585 0.610 0.071 0.337 M 4 20dehydratase mTP = probability that the sequence is mitochondrialtargeted TPlen = length of the predicted N-terminal presequence; forexample, cleavage site is predicted to be after 22aa's for ilv5.

A series of experiments were undertaken to evaluate the intracellularlocation of the wild-type ILV pathway enzymes and their subsequenttruncated counterparts. Two sets of deletions were made: one with a 6×His-tag (for localization/identification purposes), and a second setwithout one (to be used for functionality and complementationexperiments). Truncations were created with primers designed to give anN-terminal amino acid deletion and where applicable a C-terminal 6×His-tag.

Example 4 Protocols Used for Spheroplast Treatment, Fractionation, andImmunoblot Spheroplast Treatment

Spheroplast treatment was accomplished by following the manufacturer'sinstructions. A 500 ml culture of the selected yeast cell line was grownto an OD₆₀₀=1.2-1.8 in SD-Leucine selective liquid media. Cells wereharvested by centrifugation at 3000×g for 5 minutes. Two water washesfollowed with centrifugation steps—3000×g for 5 minutes. The pelletweight was determined. The cell pellet was then resuspended in 1.4ml/gram wet weight of TE Buffer, pH 8.0. The final volume of theresuspended cells was brought to 3.5 ml/gram wet weight withsterile-filtered Mill-Q water. Next 17.5 μl of β-Mercapto-ethanol pergram wet weight was added to the mixture to remove the mannan layer. Thecell mixture was incubated at 30° C. with gentle shaking on an orbitalshaker for 15 minutes. Following mannan removal, the cell mixture wascentrifuged at 3000×g for 5 minutes at room temperature. The cell pelletwas then resuspended in 4.0 ml of S-Buffer (1.0M Sorbitol, 10 mM PIPES,pH 6.5)/gram wet weight and centrifuged again at 3000×g for 5 minutes.Next, the cell pellet was resuspended again in 4.0 ml S-Buffer/gram wetweight, with the addition of 50 U of Zymolyase to remove the cell wall.This cell mixture was incubated for 60 minutes at 30° C. with gentleshaking on an orbital shaker. Post Zymolyase activity, the spheroplastswere harvested by centrifuging at 3000×g for 5 minutes at 4° C.Spheroplasts were then resuspended in 2.0 ml S-Buffer per gram wetweight and centrifuged again at 3000×g for 5 minutes. This step wasrepeated for two washes, and the final pellet was resuspended in 20 mlS-Buffer ready fro fractionation.

Fractionation

Cells were lysed via MICROFLUIDIZER at 1200 psi (Microfluidics Inc.).The cell volume was passed through the microfluidizer 5 times with restperiods on ice for 1 minute between passes. A diluted sample of cellswas checked under the microscope to ensure at least 80% lysis. Three 100μl samples of this mixture were saved at labeled “crude extract.” Thecrude extract was then centrifuged at 1000×g for 5 minutes at 4° C. Thesupernatant was transferred into a new sterile centrifuge tube andcentrifuged again at 13,000×g in a JA-20000 rotor for 10 minutes at 4°C. The pellet from the first (1000×g) spin was resuspended in 1.0 ml ofTris Buffer, pH 7.5, and three 300 μl aliquots were saved. Once thesecond (13,000×g) spin finished, the supernatant was transferred to anew sterile tube and the pellet was resuspended in 1.0 ml of TrisBuffer, pH 7.5. Three 1.0 ml aliquots of the supernatant were saved andlabeled cytosolic fraction. Three 300 μl aliquots of the resuspendedpellet were saved and labeled mitochondrial fraction. Proteinconcentrations of each fraction were determined via BCA assay kit(Thermo Scientific Inc).

Western Blot Analysis

A 4-12% Bis-Tris SDS-PAGE gel (Invitrogen Inc.) was run, with a totalprotein concentration of 7.0 ug of each fraction. The gel was run in1×MES buffer for 35 minutes. One gel was saved to Coomassie stain, andthe second was transferred to a PVDF membrane via iBlot system(Invitrogen Inc.). Detection of his-tagged protein was accomplished viaWestern Breeze chemiluminescent kit (Invitrogen Inc.). The primaryantibody was an anti-his (C-term)/AP Ab used according to manufacturer'srecommendation=1:2000 dilution for 2 hr at room temperature(cat#46-0284; Invitrogen Inc).

FIG. 22 A-D shows the results of this work.

The functional activity of these putative cytoplasmic variants of theSaccharomyces pathway was demonstrated by complementation of deletionstrains.

TABLE 3 Complementation of Deletion Strains for Isoleucine Pathway Hoststrain Complementation Complementation Strain Genotype Plasmid Genesource on SD-ILe on 5FOA number Mata his3Δ1 p415Tefllv1 S. cerevisiae+++ 7683 leu2Δ0 p415Tefllv1-Fbr S. cerevisiae +++ 7748 met15Δ0p415Tefilv1Δ25 S. cerevisiae +++ 7757 ura3Δ0 p415Tefilv1Δ35 S.cerevisiae ++ 7684 ilv1::KanMX p415Tefilv1Δ45 S. cerevisiae ++ 7685p415Tefllv1- S. cerevisiae ++ 7934 FbrΔ45 p415TefilvA C. glutamicum − −(c. opt) p415TefilvA-Fbr E.coli ++ 7510 p415TefilvA E. coli ++ −p415TefCimA L. interrogans − − (Kozak) p415Tefilv1fbr P. stipitis +++ −Mata his3Δ1 p415Tefilv5 full S. cerevisiae ++++ − leu2Δ0 p415Tefilv5Δ15S. cerevisiae ++++ − met15Δ0 p415Tefilv5Δ25 S. cerevisiae + − ura3Δ0p415Tefilv5Δ35 S. cerevisiae − − ilv5::KanMX p415Tefilv5Δ45 S.cerevisiae − − p416Tefilv5 p415Tefilv5Δ35 P. stipitis +

A similar experiment was carried out with analogous genes from Pichiastipidis the constructs shown in the below. The results are provided inFIG. 23A-D.

Strain Strain Description number S7209 p416TEF 7209 B p415TEF ilvB (Ec)7302 B + N p415TEF ilvB (Ec) p414TEFilvN (Ec) 8129 G p413TEF ilvG′ (Ec)p415TEF 7307 G + M p415TEF ilvG′(Ec) p416TEFilvM(Ec) 7558 I p415TEFilvI(Ec) 7560 I + H p415TEF ilvI(Ec) p416TEF ilvH(Ec) 7559 S ce2 p414TEFILV2 p415TEF 7309 2 + 6 p415 TEF ILV6 p414TEF ILV2 7313 B (C glu)p415TEF ilvB (Cg) 7306

Example 5 Identification/Evolution of a Keto Acid Decarboxylase forIncreased 2-Methylbutanol Production

Pyruvate, 2-ketobutyrate and 2-keto-3-methylvalerate are the threecritical keto acids whose fate is linked to the amount of 2-MBOproduction. All these three keto acids can be converted to theirrespective aldehydes by a decarboxylation reaction performed by a ketoacid decarboxylase.

In order to channel the metabolic flux towards maximum 2-MBO production,the keto acid decarboxylase should have high K_(m) for pyruvate and2-ketobutyrate, and a very low K_(m) for 2-keto-3-methylvalerate. A highK_(m) for pyruvate would prevent conversion of excess pyruvate toacetaldehyde and subsequently ethanol. Concomitantly, a high K_(m) for2ketobutyrate would prevent production of excess propanaldehyde andsubsequently propanol.

Saccharomyces cerevisiae possesses several genes encoding keto aciddecarboxylases. These enzymes have been listed as PDC1, PDC5 and PDC6.PDC1 is the major of the three decarboxylase isozymes and is involved inethanolic fermentation. Transcription of the other isoforms is glucoseand ethanol dependent. PDC1 and PDC5 expression may also be controlledby autoregulation.

PDCs from heterologous sources have been reported for high affinity forbranched keto acids. KdcA of Lactococcus lactis is the most prominentbranched chain keto acid decarboxylase (Gocke et al, 2007; Berthold etal, 2007). The activity of this enzyme, when expressed in S. cerevisiae,has however not been described earlier. Other unexplored heterologousPDCs include those of the xylose fermenting yeast Pichia stipitis (PDC1,PDC2 and PDC3(6).

The Lactococcus lactis KdcA and S. cerevisiae and P. stipitis PDCs werecloned into yeast expression vector p415 under the control of TEFpromoter. The host strain was a PDC1 deletion strain. Crude extractswere prepared for enzymatic assays by incubating cell pellet withCelLytic (Sigma), followed by bead beating. The decarboxylase reactionwas performed in 100 mM Citrate Phosphate buffer with 1 mM Thiaminediphosphate, 1 mM MgCl₂ and 50 μM to 20 mM aldehyde. The assay wascoupled to an alcohol dehydrogenase which enabled continuous monitoringof NADH or NADPH oxidation at 340 nm.

The substrate specificity of the crude extract overexpressing differentPDC/Kdcs was tested with a broad range of aldehydes. This includedacetaldehyde, butyraldehyde and 2-methylbutyraldehyde. The concentrationof the aldehydes was varied from 50M to 20 mM to determine the K_(m) andV_(max) values.

Example 6 Decarboxylase Activity on 2-keto-3-methylvalerate

2-keto-3-methylvalerate (2K3MV) was tested for decarboxylation andsubsequent reduction using PDC/KDC-ADH6 coupled assays. The results areshown in FIG. 24 (a-f). The assays show that P. stipitis PDC3/6 has thelowest K_(m) (0.84 mM) for 2-keto-3-methylvalerate. This indicates thatP. stipitis PDC 3/6 is functional in yeast and an important candidategene for increased 2-methylbutanol production. The other enzyme with lowK_(m) for 2-keto-3-methylvalerate is KDCA from L. lactis with a K_(m) of3.7 mM. However, the V_(max) value of Pichia PDC3-6 is 3.4 fold higherthan KdcA indicating that it is the most active enzyme on2-keto-3-methylvalerate.

Decarboxylase Activity on 2-Keto Butyrate

NADH dependent coupled assays with yeast ADH1 were carried out todetermine the decarboxylase activity of yeast and heterologous enzymeson the substrate 2 keto butyrate (2 KB). 2 KB is an intermediate in theproduction of 2-methylbutanol. A high decarboxylase activity on 2 KB canlead to its conversion to propanal and reduce the yield of 2-MBO. Hence,a decarboxylase with a high K_(m) for 2-ketobutyrate is of high value.Enzyme assays show that L. lactis KDCA, Pichia stipitis PDC3-6 and theKdc-PDC3/6 fusion protein KPK are equivalent in their affinity for2-ketobutyrate (See FIG. 25 a-f). The enzyme with the lowest K_(m) for2-ketobutyrate is yeast PDC1 and therefore the corresponding gene needsto be deleted in the 2-MBO production strain (data not shown). The Vmaxvalue of PDC3/6 is about 3.4 fold higher than KDCA. However, the Kms ofthe two enzymes are relatively close which suggests equal affinity for 2keto butyrate.

Decarboxylase Activity on Pyruvate

Pyruvate is the target for multiple enzymes and a decarboxylation of thecompound can lead to the production of acetaldehyde and subsequentlyethanol. To prevent decarboxylation of pyruvate and increase 2-MBOproduction, a keto acid decarboxylase with a very high K_(m) forpyruvate is ideal. Hence, all the enzymes overexpressed in S. cerevisiaewere tested for their activity with pyruvate as substrate. The kineticcurves obtained with pyruvate as substrate were sigmoidal. This could bedue to the fact that there are multiple enzymes in the crude extractwith different K_(m) for pyruvate. The final curve is therefore thesuperposition of individual curves. Absolute K_(m) values couldtherefore to be calculated (FIG. 26).

The Km and Vmax values for different enzymes and substrates aresummarized below.

Km (mM) Vmax (uM/min/mg) 2 keto 2keto 3methyl 2 keto 3methyl 2 ketoStrain valerate butyrate valerate butyrate Pyruvate ΔPDC1 5.263 6.741834.3 4924.0 8189 L. lactis 3.749 6.054 987.3 2053.0 10069 KDCA P.stipitis 0.8399 6.5090 3359. 5389 12624 PDC 3/6 KPK 8.563 4.909 819.1934 6880 (KDCA- PDC3/6 fusion)

From the above results, it can be concluded that either Lactococcuslactis KdcA or PDC3-6 from Pichia stipitis are the most suitable ketoacid decarboxylases for increased 2-MBO production. Pichia stipitisPDC3/6 is however a superior enzyme for decarboxylation of branched ketoacids.

Example 7 Identification of an Alcohol Dehydrogenase (ADH) with HighAffinity for Branched Aldehydes

The genome of S. cerevisiae shows the presence of 7 different alcoholdehydrogenases (ADHs). The ideal ADH for 2-MBO production should be ableto reduce branched aldehydes. Therefore, the enzymes ADH1, ADH6, andADH7 were overexpressed in yeast and were tested for their reductaseactivity on various aldehydes. Yeast ADH1, which is also commerciallyavailable was very active on acetaldehyde and butyraldehyde, but was˜5000 fold less active on 2-methyl butaraldehyde than acetaldehyde (FIG.27). This suggested that ADH1 is not an ideal enzyme in the 2-MBOproduction pathway.

Yeast ADH6 and ADH7 have been reported as broad substrate specificityalcohol dehydrogenases. These enzymes were therefore overexpressed in S.cerevisiae under the control of TEF promoter and enzyme assays werecarried out with crude extracts. Both ADH6 and ADH7 were active on2-methylbutanal and were NADPH dependent reductases.

ADH6 showed higher affinity for branched aldehyde and was subsequentlycloned as a histidine tagged protein in the yeast expression vectorp415TEF and purified by affinity chromatography.

Enzyme assays for aldehyde reductase activity were carried out in 100 mMcitrate phosphate buffer pH6.2, 0.5 mM NADPH and 0.05 to 20 mM substrate(acetaldehyde or 2-methylbutanal). The reaction was monitored for 5minutes at 340 nm for oxidation of NADPH to NADP+. Results are shown inFIG. 28.

The pure enzyme showed Michaelis Menten kinetics for both thesubstrates. The K_(m) and V_(max) values were determined for bothacetaldehyde and 2-methyl butyraldehyde and are summarized in FIG. 29.

As evident from the above figures, yeast ADH6 shows a lower K_(m) (0.36mM) for 2-methyl butyraldehyde than acetaldehyde (0.58 m). The V_(max)value (cofactor oxidation rate) for 2-methyl butyraldehyde is also 1.3fold higher than acetaldehyde (FIGS. 30 and 31).

ADH6 was therefore identified as the ideal enzyme for 2-MBO productiondue to its ability to perform reduction of 2-methyl butyraldehyde to2-methylbutanol.

Example 8 Pyruvate (PDC) and keto-acid decarboxylase (Kdc) Screening

Genes screened for increased production of 2-MBO included: PDC1 (S.cerevisiae), PDC5 (S. cerevisiae), PDC6 (S. cerevisiae), THI3 (S.cerevisiae), PDC1 (P. stipitis), PDC2 (P. stipitis), PDC3-6 (P.stipitis), KivD (L. lactis), KdcA (L. lactis), KdcA-S286Y (L. lactis),and Kdc (M. tuberculosis).

The aim of the characterization of these genes was to distinguishdecarboxylases with a low Km for 2-keto-3-methylvalerate and a high Kmfor pyruvate.

Bioinformatic and structural analysis show structure similarities topyruvate decarboxylase and keto acid decarboxylases. Amino acid andconserved domain analysis showed the potential for mutation or switchingactive sites with the possibility of turning a PDC into a Kdc, therebyaltering the affinity of Saccharomyces PDC for pyruvate and other ketoacids and forcing the conversion of 2-keto-3-methylvalerate to the 2methyl-butyraldehyde.

PDC1 Mutant Library

Pdc1p (S. cerevisiae) was aligned with KdcAp (L. lactis) to determine ifthe same sites altered in KdcAp to increase affinity for2-keto-3-methyl-valerate could be saturated in Pdc1p via degenerateprimers to (FIG. 32). Primers were designed to produce various mutants,as exemplified in FIG. 33. Exemplary exchange sites were selected, asillustrated in FIGS. 34 and 35. Exemplary constructions were generatedas shown in FIGS. 36-38.

These mutant libraries were transformed into yeast and tested inhigh-throughput style for increased levels of 2-MBO when fed threonineat 20 mM.

Fusion Proteins

To attain the strong characteristics of PDC1 and KDCA, high activity inS. cerevisiae and high affinity towards 2-keto-3-methyl-valerate,respectively, different protein fusions were created linking the twoproteins at different domain locations. Images of the domains weredownloaded from NCBI and sites were chosen by visualizing the crystalstructure of both Pdclp and KdcAp.

TABLE 4 This 2-MBO Production in the PDC1Δ background. Data shows thatKdcAp from Lactococcus lactis is better for final MBO production SGI #Full Name 3-MBO 2-MBO 7658 ΔPDC1: p415TEF-Kdc (Mt) 98.69 61.42 98.3761.79 100.10 66.77 7659 ΔPDC1: p415TEF-KdcA(L1) 183.94 170.07 187.04181.92 190.16 190.95 7660 ΔPDC1: p415TEF-PDC1 106.13 84.49 101.55 79.94106.81 91.68

Additional fusion proteins between L. lactis KDCA and P. stipitis PDC3-6were also made as shown in FIG. 39-41.

Example 9 Evaluation of Transcripts (mRNA) by Semi-Quantitative PCR

Method for RNA Extraction from Yeast Cells Using Glass Beads

2.0 ml tubes containing 0.25 g acid washed glass beads (0.5 mm diameter)and 250 μl Phenol:CHCl₃:isoamyl alcohol (25:24:1) were prepared inadvance and placed on ice. Yeast was collected at 5 OD₆₀₀ units (e.g.,10 ml of OD₆₀₀ 0.5). Cells were spun at 4° C. for 5 minutes at 2000 rpm,then resuspended in 2 ml of ice cold DEPC-treated HE (10 mM HEPES, 1 mMEDTA, pH 8) and transferred to microfuge tubes. Cells were then spun asecond time at 4° C. for 30 seconds at top speed. Pellets were stored onice for immediate use. Cells were resuspended a second time in 250 μlHENS buffer (10 mM HEPES-NaOH, pH 7.5 Treat with DEPC, 1 mM EDTA, 300 mMNaCl, and 0.2% SDS) and quickly transferred to 2.0 ml tubes with glassbeads. Cells were then vortexed for 10 seconds, doing one tube at a timeand placed on ice. All tubes were then vortexed at 4° C. for 30 minutes(25-75% breakage). The tubes were then spun for 30 minutes at 4° C. attop speed. 200 μl of the supernatant was transferred to a 1.5 mlmicrofuge tube without collecting any of the interface. Extraction wasrepeated adding an equal volume Phenol:CHCl₃: isoamyl alcohol, thenvortexed for 15 seconds and spun for 5 minutes at top speed. Threevolumes of 100% EtOH was added to the supernatant then mixed thoroughly.Samples were precipitated overnight at −20° C. Samples were then spun at4° C. for 30 minutes at top speed. Supernatant was carefully removed andpellets were washed with 150 μl of 75% EtOH (DEPC treated). Pellets werespun again for 5 minutes at top speed, supernatant was carefullyremoved. Samples were dried in Speed-Vac and resuspended in 10 μlDEPC-treated ddH2O per 1 OD600 unit. RNA solution was kept on ice oncepellet was dissolved and used immediately with the INVITROGENSUPERSCRIPT III REVERSE TRANSCRIPTASE KIT. RNA was tested for equalconcentration prior to cDNA production via equal loading on an RNAdenaturation gel. RNA was stored at −20° C. for a maximum of one week.

TABLE 5 Primers used for RNA Expression Analysis of Specific Yeast GenesPrimer # Gene Amplified Primer SEQ ID NO 283 KdcA Forward Primer5′-atgtatactgtgggggattatttgttggat-3′ SEQ ID NO: 91 284 KdcA ReversePrimer 5′-ttatttgttttgctcagcaaatagtttccc-3′ SEQ ID NO: 92 370 ACT1Forward Primer 5′-atggattctgaggttgctgctt-3′ SEQ ID NO: 93 371 ACT1Reverse Primer 5′-ttagaaacacttgtggtgaacgatag-3′ SEQ ID NO: 94 372 ADH6Forward Primer 5′-atgtcttatcctgagaaatttgaaggta-3′ SEQ ID NO: 95 373 ADH6Reverse Primer 5′-ctagtctgaaaattctttgtcgtag-3′ SEQ ID NO: 96

The synthetic SGI YACv1.0 was created by Isothermal assembly reactionusing individual gene cassettes, and also truncated-hybrid cassettes.The individual gene cassette method is first described. The followinggenes were previously subcloned into p4xx series yeast plasmids. Allplasmids contained the TEF promoter and CYC terminator.Promoter-ORF-Terminator cassettes were amplified from plasmids withprimers to create 40 base overlaps to either tandem cassettes or pYAC4EcoRI flanking bases (See FIG. 42).

Example 10 Individual Gene Cassette Method

Six cassettes were amplified and gel purified, then mixed together withthe pYAC4 digested with EcoRI and gel purified in equal molar ratios andassembled by Isothermal Assembly method. The genes included in eachcassette are listed below.

Cassette Gene Source Organism 1 ilvA^(FBR) E. coli 2 ilvG E. coli 3 ilvCE. coli 4 ilvD E. coli 5 kdcA L. lactis 6 ADH6 S. cerevisiae

The Isothermal Assembly Reaction was setup in 201 reactions. The finalconcentrations were: FINAL 80 μl mixture; containing 100 mM Tris-Cl pH7.5), 10 mM MgCl₂, 200 μM dGTP, 200 μM dATP, 200 μM dTTP, 200 μM dCTP,10 mM DTT, 5% PEG-8000, 1 mM NAD, 0.004 U μl⁻¹ T5 exonuclease(Epicentre), 0.025 U μl⁻¹ Phusion polymerase (NEB), 4 U μl Taq Ligase(NEB), and DNA mixture ˜40 ng per kb.

The reaction was left at 50° C. for 1 hr, then phenol chloroformextracted, and NaCl precipitated. It was resuspended in 20 μl H₂O, andtransformed into S. cerevisiae ATCC 200897 strain (MATα ade2Δ::hisGhis3Δ200 leu2Δ0 met15Δ0 trp1Δ63 ura3Δ0) and selected on SD-ura-trp agarplates with 20 mg/L of adenine for red and white colony selection inpYAC4. This is because the Sup-4-o suppressor tRNAgene has the EcoRIsite in the middle, and this was where we inserted our cassettes. ColonyPCR (RedTaq) was used to screen 96 colonies. Primers were designed toamplify a 1.6 kb band from the middle of ilvC to the middle of ilvD. Thehit was then streaked out, and the new circular YAC was purified usingthe Yeast Plasmid Prep II kit (Zymo Research). This was transformed intoSTBL4 cells (Invitrogen), and 3 colonies were grown and sent tosequencing. The preps were put through PCR profiling that spanned acrossthe construct from the middle of one gene to the middle of the nexttandem gene (data not shown).

Plasmid preps from 3 different STBL4 transformants as well as the emptyvector were put into 8 different PCR reactions each. A=1.6 kb amplifiedfrom −400 bases upstream of the EcoRI site in pYAC4 to the middle ofilvA.B=2.5 kb amplified from the middle of ilvA to the middle ofilvG.C=2.7 kb amplified from the middle of ilvG to the middle ofilvC.D=1.8 kb amplified from the middle of ilvC to the middle ofilvD.E=2.3 kb amplified from the middle of ilvD to the middle ofkdcA.F=2.4 kb amplified from the middle of kdcA to the middle ofadh6.G=1.3 kb amplified from the middle of adh6 to +400 bases downstreamof the EcoRI site in pYAC4.H=4.1 kb amplified from middle of ilvC to themiddle of kdcA. This spans the ilvD, as well as two repeating TEF-CYCelements, thus the prevalent 1.5 kb band is amplified because thetemplate loops large TEF homologies, and the polymerase amplifies theshorter fragment without the ilvD. 4.1 kb band is seen faintly.

Example 11 Truncated-Hybrid Method

The same method as the individual cassettes, but truncated hybridcassettes have repetitive elements tucked in the middle of thecassettes, and partial genes on either side. The truncated hybridcassettes are created by amplifying the primary cassettes (FIG. 43).These cassettes are then mixed together in an equal molar ratio, andthen PCR amplified again using primers located in the middle of thetandem gene cassette. The product, the truncated-hybrid cassettes have40 base overlap homology to the next tandem truncated-hybrid cassette.The same was done for the first and last genes using vector as one ofthe overlapping primary cassettes. Six primary cassettes and vector endswere used to create 7 truncated-hybrid cassettes. These bands were thengel purified and mixed together in equal molar ratios.

Then 3 fragments were stitched together with Isothermal Assembly tocreate 1 larger fragment of 7 kb (FIG. 44), and the remaining 4fragments were stitched together to create 1 larger fragment of 8 kb(FIG. 45). The Isothermal conditions were the same as described above,however this time reactions were setup with 400 ng/kb and left at 50 Cfor 3 hours. The entire reaction was loaded onto a 0.8% agarose gel andrun for 1 hour at 60V, and gel purified using ZymoClean kit. These weremixed with the linearized vector and stitched together with IsothermalAssembly (100 ng/kb, 50 C 3 hrs), and transformed into S. cerevisiaeATCC 200897 strain. In parallel, the 3 final fragments were alsotransformed directly into S. cerevisiae to allow for in-vivorecombination of the fragments into a circular molecule (FIG. 46). Hitswere screened the same way as the “Individual Gene Cassette Method”described above and sent to be sequenced. The STBL4 grown plasmids weresequenced by BATJ with 22 primers. The table below represents an entireinsert sequenced from the invitro recombination method using the 3fragments.

Example 12 Truncated-Hybrid Method to Create 16 Gene YAC

Used the same method as the truncated-hybrid method to create 6 geneYAC. 16 primary gene cassettes were amplified, then Overlap PCR was usedto create the truncated-hybrid cassettes. All genes had TEF promoter,CYC terminator. The genes used were:

Gene Source Organism PCK P. stipitis AAT2 P. stipitis HOM3^(FBR) P.stipitis HOM2 P. stipitis HOM6 P. stipitis THR1 P. stipitis THR4 P.stipitis ilvA^(FBR) E. coli ilvG E. coli ilvM E. coli ilvC E. coli ilvDE. coli kdcA L. lactis ADH6 S. cerevisiae

Three 3 sets of 4 fragments with overlap to each other, and 1 set of 3fragments with overlap to each other, were stitched together withIsothermal Assembly to create larger intermediate fragments, similar tothe methods described above, but with 16 genes total and 4 intermediatefragments. The Isothermal conditions were the same as described above,however this time reactions were setup with 400 ng/kb and left at 50 Cfor 6 hours. The entire reaction was loaded onto a 0.8% agarose gel andrun for 1 hour at 70V, and gel purified using ZymoClean kit. These weremixed with the linearized vector (20 ng/kb each fragment) andtransformed into S. cerevisiae ATCC 200897 strain to allow for in-vivorecombination of the fragments into a circular molecule. Colonies werescreened using primers to amplify across junctions of recombinedintermediate fragments. The presumed insert sequence of the entireinsert for 16 gene YAC is provided below (SEQ ID NO:97).

atagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaatttcccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaaattttttttttgatttttttctctttcgatgacctcccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaagttttctagaactagtggatcccccatggctgactcgcaacccctgtccggtgctccggaaggtgccgaatatttaagagcagtgctgcgcgcgccggtttacgaggcggcgcaggttacgccgctacaaaaaatggaaaaactgtcgtcgcgtcttgataacgtcattctggtgaagcgcgaagatcgccagccagtgcacagctttaagctgcgcggcgcatacgccatgatggcgggcctgacggaagaacagaaagcgcacggcgtgatcactgcttctgcgggtaaccacgcgcagggcgtcgcgttttcttctgcgcggttaggcgtgaaggccctgatcgttatgccaaccgccaccgccgacatcaaagtcgacgcggtgcgcggcttcggcggcgaagtgctgctccacggcgcgaactttgatgaagcgaaagccaaagcgatcgaactgtcacagcagcaggggttcacctgggtgccgccgttcgaccatccgatggtgattgccgggcaaggcacgctggcgctggaactgctccagcaggacgcccatctcgaccgcgtatttgtgccagtcggcggcggcggtctggctgctggcgtggcggtgctgatcaaacaactgatgccgcaaatcaaagtgatcgccgtagaagcggaagactccgcctgcctgaaagcagcgctggatgcgggtcatccggttgatctgccgcgcgtagggctatttgctgaaggcgtagcggtaaaacgcatcggtgacgaaaccttccgtttatgccaggagtatctcgacgacatcatcaccgtcgatagcgatgcgatctgtgcggcgatgaaggatttattcgaagatgtgcgcgcggtggcggaaccctctggcgcgctggcgctggcgggaatgaaaaaatatatcgccctgcacaacattcgcggcgaacggctggcgcatattctttccggtgccaacgtgaacttccacggcctgcgctacgtctcagaacgctgcgaactgggcgaacagcgtgaagcgttgttggcggtgaccattccggaagaaaaaggcagcttcctcaaattctgccaactgcttggcgggcgttcggtcaccgagttcaactaccgttttgccgatgccaaaaacgcctgcatctttgtcggtgtgcgcctgagccgcggcctcgaagagcgcaaagaaattttgcagatgctcaacgacggcggctacagcgtggttgatctctccgacgacgaaatggcgaagctacacgtgcgctatatggtcggcggacgtccatcgcatccgttgcaggaacgcctctacagcttcgaattcccggaatcaccgggcgcgctgctgcgcttcctcaacacgctgggtacgtactggaacatttctttgttccactatcacagccatggcaccgactacgggcgcgtactggcggcgttcgaacttggcgaccatgaaccggatttcgaaacccggctgaatgagctgggctacgattgccacgacgaaaccaataacccggcgttcaggttctttttggcgggttagggggctgcaggaattcgatatcaagcttatcgataccgtcgacctcgagtcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcggccggtacccaattcgccctatagtgagtcgtattacgcgcgcatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaatttcccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaaattttttttttgatttttttctctttcgatgacctcccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaagttttctagaactagtggatcccccatgaatggcgcacagtgggtggtacatgcgttgcgggcacagggtgtgaacaccgttttcggttatccgggtggcgcaattatgccggtttacgatgcattgtatgacggcggcgtggagcacttgctatgccgacatgagcagggtgcggcaatggcggctatcggttatgctcgtgctaccggcaaaactggcgtatgtatcgccacgtctggtccgggcgcaaccaacctgataaccgggcttgcggacgcactgttagattccatccctgttgttgccatcaccggtcaagtgtccgcaccgtttatcggcactgacgcatttcaggaagtggatgtcctgggattgtcgttagcctgtaccaagcacagctttctggtgcagtcgctggaagagttgccgcgcatcatggctgaagcattcgacgttgcctgctcaggtcgtcctggtccggttctggtcgatatcccaaaagatatccagttagccagcggtgacctggaaccgtggttcaccaccgttgaaaacgaagtgactttcccacatgccgaagttgagcaagcgcgccagatgctggcaaaagcgcaaaaaccgatgctgtacgttggcggtggcgttggtatggcgcaggcagttccggctttgcgtgaatttctcgctgccacaaaaatgcctgccacctgtacgctgaaagggctgggcgcagtagaagcagattatccgtactatctgggcatgctgggaatgcatggcaccaaagcggcgaacttcgcggtgcaggagtgcgacttgctgatcgccgtgggtgcacgttttgatgaccgggtgaccggcaaactgaacaccttcgcaccacacgccagtgttatccatatggatatcgacccggcagaaatgaacaagctgcgtcaggcacatgtggcattacaaggtgatttaaatgctctgttaccagcattacagcagccgttaaatatcaatgactggcagctacactgcgcgcagctgcgtgatgaacatgcctggcgttacgaccatcccggtgacgctatctacgcgccgttgttgttaaaacaactgtcagatcgtaaacctgcggattgcgtcgtgaccacagatgtggggcagcaccagatgtgggctgcgcagcacatcgcccacactcgcccggaaaatttcatcacctccagcggcttaggcaccatgggttttggtttaccggcggcggttggcgcgcaagtcgcgcgaccaaacgataccgtcgtctgtatctccggtgacggctctttcatgatgaatgtgcaagagctgggcaccgtaaaacgcaagcagttaccgttgaaaatcgtcttactcgataaccaacggttagggatggttcgacaatggcagcaactgtttttccaggaacgatatagcgaaaccacccttaccgataaccccgatttcctcatgttagccagcgccttcggcatccctggccaacacatcacccgtaaagaccaggttgaagcggcactcgacaccatgctgaacagtgatgggccatacctgcttcatgtctcaatcgacgaacttgagaacgtctggccgctggtgccgcctggtgccagtaattcagaaatgttggagaaattatcatgatgcaacatcaggtcaatgtatcggctctgagggctgcaggaattcgatatcaagcttatcgataccgtcgacctcgagtcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcggcctcactggccgtcgttttacaacgtcgtgactgggaaaaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaatttcccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaaattttttttttgatttttttctctttcgatgacctcccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaagttttctagaactagtggatcccccatggctaactacttcaatacactgaatctgcgccaacagctggcacagctgggcaaatgtcgctttatgggccgcgatgaattcgccgatggcgcgagctaccttcagggtaaaaaagtagtcatcgtcggctgtggcgcacagggtctgaaccagggcctgaacatgcgtgattctggtctcgatatctcctacgctctgcgtaaagaagcgattgccgagaagcgcgcgtcctggcgtaaagcgaccgaaaatggttttaaagtgggtacttacgaagaactgatcccacaggcggatctggtgattaacctgacgccggacaagcagcactctgatgtagtgcgcaccgtacagccactgatgaaagacggcgcggcgctgggctactcgcacggtttcaacatcgtcgaagtgggcgagcagatccgtaaagatatcaccgtagtgatggttgcgccgaaatgcccaggcaccgaagtgcgtgaagagtacaaacgtgggttcggcgtaccgacgctgattgcccttcacccggaaaacgatccgaaaggcgaaggcatggcgattgccaaagcctgggcggctgcaaccggtggtcaccgtgcgggtgtgctggaatcgtccttcgttgcggaagtgaaatctgacctgatgggcgagcaaaccatcctgtgcggtatgttgcaggctggctctctgctgtgcttcgacaagctggtggaagaaggtaccgatccagcatacgcagaaaaactgattcagttcggttgggaaaccatcaccgaagcactgaaacagggcggcatcaccctgatgatggaccgtctctctaacccggcgaaactgcgtgcttatgcgctttctgaacagctgaaagagatcatggcacccctgttccagaaacatatggacgacatcatctccggcgaattctcttccggtatgatggcggactgggccaacgatgataagaaactgctgacctggcgtgaagagaccggcaaaaccgcgtttgaaaccgcgccgcagtatgaaggcaaaatcggcgagcaggagtacttcgataaaggcgtactgatgattgcgatggtgaaagcgggcgttgaactggcgttcgaaaccatggtcgattccggcatcattgaagagtctgcatattatgaatcactgcacgagctgccgctgattgccaacaccatcgcccgtaagcgtttgtacgaaatgaacgtggttatctctgataccgctgagtacggtaactatctgttctcttacgcttgtgtgccgttgctgaaaccgtttatggcagagctgcaaccgggcgacctgggtaaagctattccggaaggcgcggtagataacgggcaactgcgtgatgtgaacgaagcgattcgcagccatgcgattgagcaggtaggtaagaaactgcgcggctatatgacagatatgaaacgtattgctgttgcgggttaagtgggctgcaggaattcgatatcaagcttatcgataccgtcgacctcgagtcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcggccctggcgttacccaacttaatcgccttgcagcacatcccccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaatttcccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaaattttttttttgatttttttctctttcgatgacctcccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaagttttctagaactagtggatcccccatgcctaagtaccgttccgccaccaccactcatggtcgtaatatggcgggtgctcgtgcgctgtggcgcgccaccggaatgaccgacgccgatttcggtaagccgattatcgcggttgtgaactcgttcacccaatttgtaccgggtcacgtccatctgcgcgatctcggtaaactggtcgccgaacaaattgaagcggctggcggcgttgccaaagagttcaacaccattgcggtggatgatgggattgccatgggccacggggggatgctttattcactgccatctcgcgaactgatcgctgattccgttgagtatatggtcaacgcccactgcgccgacgccatggtctgcatctctaactgcgacaaaatcaccccggggatgctgatggcttccctgcgcctgaatattccggtgatctttgtttccggcggcccgatggaggccgggaaaaccaaactttccgatcagatcatcaagctcgatctggttgatgcgatgatccagggcgcagacccgaaagtatctgactcccagagcgatcaggttgaacgttccgcgtgtccgacctgcggttcctgctccgggatgtttaccgctaactcaatgaactgcctgaccgaagcgctgggcctgtcgcagccgggcaacggctcgctgctggcaacccacgccgaccgtaagcagctgttccttaatgctggtaaacgcattgttgaattgaccaaacgttattacgagcaaaacgacgaaagtgcactgccgcgtaatatcgccagtaaggcggcgtttgaaaacgccatgacgctggatatcgcgatgggtggatcgactaacaccgtacttcacctgctggcggcggcgcaggaagcggaaatcgacttcaccatgagtgatatcgataagctttcccgcaaggttccacagctgtgtaaagttgcgccgagcacccagaaataccatatggaagatgttcaccgtgctggtggtgttatcggtattctcggcgaactggatcgcgcggggttactgaaccgtgatgtgaaaaacgtacttggcctgacgttgccgcaaacgctggaacaatacgacgttatgctgacccaggatgacgcggtaaaaaatatgttccgcgcaggtcctgcaggcattcgtaccacacaggcattctcgcaagattgccgttgggatacgctggacgacgatcgcgccaatggctgtatccgctcgctggaacacgcctacagcaaagacggcggcctggcggtgctctacggtaactttgcggaaaacggctgcatcgtgaaaacggcaggcgtcgatgacagcatcctcaaattcaccggcccggcgaaagtgtacgaaagccaggacgatgcggtagaagcgattctcggcggtaaagttgtcgccggagatgtggtagtaattcgctatgaaggcccgaaaggcggtccggggatgcaggaaatgctctacccaaccagcttcctgaaatcaatgggtctcggcaaagcctgtgcgctgatcaccgacggtcgtttctctggtggcacctctggtctttccatcggccacgtctcaccggaagcggcaagcggcggcagcattggcctgattgaagatggtgacctgatcgctatcgacatcccgaaccgtggcattcagttacaggtaagcgatgccgaactggcggcgcgtcgtgaagcgcaggacgctcgaggtgacaaagcctggacgccgaaaaatcgtgaacgtcaggtctcctttgccctgcgtgcttatgccagcctggcaaccagcgccgacaaaggcgcggtgcgcgataaatcgaaactggggggttaagggctgcaggaattcgatatcaagcttatcgataccgtcgacctcgagtcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcggcctttcgccagctggcgtaatagcgaagaggccccgcaccgatatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaatttcccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaaattttttttttgatttttttctctttcgatgacctcccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaagttttctagaactagtggatcccccatgtatactgtgggggattatttgttggataggttgcatgaattaggcatcgaggaaatctttggtgtacctggagattacaatttgcaatttctggaccagatcatatcgagagaggatatgaaatggattggtaacgccaatgaattaaatgccagctatatggccgatggctatgctcgtaccaagaaagctgctgcttttctgacaacttttggtgtcggtgaattgtctgctattaacggactggccggtagttatgctgaaaatttgccagtagttgaaatagtcggaagcccaacttctaaagtgcaaaacgatggcaaattcgtgcatcatactctggcagatggtgattttaagcacttcatgaaaatgcatgaacccgtaacggctgccagaactcttttaacagccgagaatgcgacatatgaaattgatcgtgtactttctcagcttttaaaggagagaaaacctgtttacataaacttacctgtcgatgttgctgctgccaaagcagagaagccagccctgtctcttgaaaaagaaagctccaccaccaacactaccgaacaagtgatattatctaaaattgaggaatcacttaaaaacgctcagaaaccagtagtcatagcgggtcatgaagtcataagtttcggtcttgaaaagactgtaacacaatttgtcagcgaaacaaaattgcctatcactactttgaactttggcaaaagtgcggtcgacgagtcgttgccatcatttttgggtatctacaatggcaaactatcagaaatctcattgaaaaatttcgtagaaagtgcggatttcattctgatgttgggcgtcaagctgacggattcttctacgggggctttcactcaccatttggatgaaaacaaaatgatttcattgaacatcgatgaagggatcatctttaataaggtagtggaagatttcgattttagagccgtggtttcctccttatcagagttaaaaggtattgagtacgaagggcagtatattgataagcagtacgaggaatttattccttcttctgctccactttctcaagatcgtttatggcaagcagtcgagtccctgacacaaagcaacgagactatagttgcagagcaggggacctcattctttggtgcctctacaatttttctgaaatccaacagcagatttataggacaacccctttggggctctattggatatacttttcccgcagcccttggttcacaaatcgcagataaggagtcaagacatctgttattcataggtgatggtagtctacaattaacagttcaagaattaggcctatcaataagggagaagttaaacccaatctgtttcataattaacaatgacggctacactgttgaaagggagatccacggaccaacacaatcatacaatgatattcccatgtggaactatagcaaattaccggagactttcggcgcaaccgaggatagagtagtttcgaagatcgttaggactgagaatgaatttgttagcgttatgaaggaagcccaggctgatgtcaatagaatgtattggattgaattagttttggaaaaggaagatgcacctaaattactaaaaaagatggggaaactatttgctgagcaaaacaaataagggctgcaggaattcgatatcaagcttatcgataccgtcgacctcgagtcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcggcccgcccttcccaacagttgcgcagcctgaatggcgaatggcatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaatttcccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaaattttttttttgatttttttctctttcgatgacctcccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaagttttctagaactagtggatcccccatgtcttatcctgagaaatttgaaggtatcgctattcaatcacacgaagattggaaaaacccaaagaagacaaagtatgacccaaaaccattttacgatcatgacattgacattaagatcgaagcatgtggtgtctgcggtagtgatattcattgtgcagctggtcattggggcaatatgaagatgccgctagtcgttggtcatgaaatcgttggtaaagttgtcaagctagggcccaagtcaaacagtgggttgaaagtcggtcaacgtgttggtgtaggtgctcaagtcttttcatgcttggaatgtgaccgttgtaagaatgataatgaaccatactgcaccaagtttgttaccacatacagtcagccttatgaagacggctatgtgtcgcagggtggctatgcaaactacgtcagagttcatgaacattttgtggtgcctatcccagagaatattccatcacatttggctgctccactattatgtggtggtttgactgtgtactctccattggttcgtaacggttgcggtccaggtaaaaaagttggtatagttggtcttggtggtatcggcagtatgggtacattgatttccaaagccatgggggcagagacgtatgttatttctcgttcttcgagaaaaagagaagatgcaatgaagatgggcgccgatcactacattgctacattagaagaaggtgattggggtgaaaagtactttgacaccttcgacctgattgtagtctgtgcttcctcccttaccgacattgacttcaacattatgccaaaggctatgaaggttggtggtagaattgtctcaatctctataccagaacaacacgaaatgttatcgctaaagccatatggcttaaaggctgtctccatttcttacagtgctttaggttccatcaaagaattgaaccaactcttgaaattagtctctgaaaaagatatcaaaatttgggtggaaacattacctgttggtgaagccggcgtccatgaagccttcgaaaggatggaaaagggtgacgttagatatagatttaccttagtcggctacgacaaagaattttcagactaggggctgcaggaattcgatatcaagcttatcgataccgtcgacctcgagtcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcggccFermentation DataThreonine Deaminase

The conversion of L-threonine to 2-keto butyrate (2-oxobutanoate) iscatalyzed by Threonine Deaminase (TD). Two types of TD have beendescribed. The catabolic TD is expressed during the utilization ofthreonine as a nitrogen source. This enzyme, encoded by the CHA1 gene,is primarily genetically regulated. The biosynthetic TD, encoded by theILV1 gene in S. cerevisiae, catalyzes the same reaction. However, thisenzyme is subject to allosteric regulation by isoleucine. Deregulationof TD either by expression of the catabolic TD or expression of abiosynthetic TD that is insensitive to isoleucine increased productionof 2-MBO (FIG. 47).

Screening in tubes produced a similar result (1500-2000 μM 2-MBO in 24hours).

Example 13 Cytosolic TD

In yeast, the metabolic reactions in isoleucine biosynthesis fromthreonine are thought to occur predominantly in the mitochondria.Expression of isoleucine-insensitive, cytosolic TD resulted in theaccumulation of propanol. Propanol results from the decarboxylation andsubsequent reduction of 2-KB. The accumulation of propanol confirms theactivity of the recombinant or modified TD.

TABLE 5 Fermentation products during expression of cytosolic TD. StrainID Description 3-MBO¹ 2-MBO Propanol Isobutanol 8002 p415TEF ILV1^(FBR)130 102 632 1121 delta45::His 7466 p414TEF ilvA^(FBR) (Gg) 78 36 522 233p415TEF 7511 p416Tef ilvA^(FBR) (Ec) 75 52 1855 373 p415TEF N/A Controls73 125 317 514 ¹All concentrations are in μM.

Example 14 Acetolactate Synthase (Acetohydroxy Acid Synthase, ALS)

The first committed step in Valine/Leucine biosynthesis and Isoleucinebiosynthesis is catalyzed by ALS. This enzyme catalyzes the formation ofeither 2-acetolactate from two pyruvate molecules or formation of2-aceto-2-hydroxy-butyrate from 1 molecule pyruvate and 1 molecule of2-KB. The regulation of this enzymatic step is complex and itsbiochemistry in S. cerevisiae has not been well characterized.

Expression of various ALS genes alone primarily lead to increasedisobutanol production (Valine production) in tube experiments.

TABLE 6 Fermentation Products during ALS Expression Strain ID Name2-MBO¹ 3-MBO nPrOH iBuOH 7305 p415TEF ILV6 56.05 194.37 349.65 210.217306 p415TEF ilvB (Cg) 55.55 183.79 294.29 256.52 7308 p415TEF ilvB (Ec)54.35 153.11 297.14 348.00 7309 p414TEF ilv2 + p415TEF 53.44 187.23309.37 506.19 7558 p415TEF ilvG′ (Ec) p416TEF ilvM(Ec) 19.59 32.39140.09 111.81 7559 p415TEF ilvI (Ec) p416TEF ilvH(Ec) 58.24 93.93 325.29377.38 7560 p415TEF ilvI(Ec) 17.47 23.17 54.78 103.03 7561 p415TEFilvG'(E) 15.54 20.36 46.35 87.06 7732 p415TEF AlsS (Bs) 48.00 67.33217.07 360.02 7733 p415TEF AlsS* (Bs) 56.19 74.99 310.28 503.28 7888p415TEF ilv2(Δ45::His) 87.91 106.90 350.70 992.34 7890 p415TEF AlsS(Bs)-AHFGQ 95.04 125.21 355.78 1039.90 7891 p415TEF AlsS (Bs)-VHFNQ86.93 142.12 318.68 796.19 7892 p415TEF AlsS (Bs)-VHFPQ 96.46 123.53362.70 1111.76 7893 p415TEF AlsS (Bs)-VHFQQ 100.83 128.40 354.80 1125.827914 p415TEF ilv2::His 86.85 128.99 342.29 1375.85 7915 p415TEF ilv2(Δ25::His) 95.57 111.87 327.92 941.70 7916 p415TEF ilv2: (Δ35::His 81.87123.46 285.78 624.58 ¹All concentrations are in μM.

Example 15 TD and ALS

Co-expression of a cytosolic TD with a cytosolic ALS decreases propanolproduction, indicating that the ALS is competing with KDC for the 2-KB(Table 7). If the subsequent enzymes in the pathway were expressed inthe cytoplasm, increased 2-MBO production would be expected.

TABLE 7 Fermentation Products during Co-Expression of TD and ALS. StrainID Name 2-MBO¹ 3-MBO nPrOH iBuOH 7511 p416TEF ilvA^(FBR) (Ec) p415TEF 5175 1854 373 7720 p415TEF ilvG′ (Ec) p413TEF-i1vA^(FBR)(Ec) 49 76 896 4977729 p415TEFilvI (Ec) p413TEF-ilvA^(FBR) (Ec) 48 74 1061 580 ¹Allconcentrations are in μM.Aspartate Kinase and TD

The first reaction in Threonine biosynthesis is catalyzed by theallosteric enzyme aspartate kinase (AK), encoded by the HOM3 gene inyeasts. The accumulation of intracellular threonine in strainsexpressing threonine-insensitive AK suggests that flux through thispathway had been increased. Expression in a strain co-expressing anisoleucine-insensitive TD increased 2-MBO specific productivity (FIG.48). Fermentation titer was further increased by expression of eitherTHR1 or THR4.

TABLE 8 Fermentation Products in TD and AK Expression Strain Genotype3-MBO 2-MBO Propanol isobutanol 8020 :TEF ILV^(FBR) p413TEF p415TEFp416CYC 118 2170 543 867 HOM3-R2 8021 :TEF ILV^(FBR) p413TEF p415TEFHOM2 125 2429 699 975 p416CYC HOM3-R2 8022 :TEF ILV^(FBR) p413TEF HOM6p415TEF 124 2414 499 856 HOM 2 p416CYC HOM3-R2 8023 :TEF ILV^(FBR)p413TEF THR1 p415TEF 132 2748 904 1068 Hom2 p416CYC HOM3-R2 8024 :TEFILV^(FBR) p413TEF THR4 p415TEF 140 2834 581 824 HOM2 p416CYC HOM3-R2)Selection of ADH

Biochemical studies determined that the conversion of 2-MBA to 2-MBO isnot effectively performed by the endogenously expressed alcoholdehydrogenases, which is presumed to be ADH1. This was evaluated in vivoby supplementing growth medium with the aldehyde precursor of 2-MBO incultures expressing various alcohol dehydrogenases.

Cultures of S. cerevisiae expressing various ADH enzymes in an otherwiseisogenic background were incubated by standard methods for 12 hours. Abolus of 2-MBA was added to the medium at 12 hours EFT. Aldehydes arenot charged and can freely diffuse into the cells. Samples werecollected hourly and 2-MBO in the fermentation broth was measured. Thespecific rate of 2-MBO production (μmol 1-1 OD600-1 h-1) was determinedduring the period of excess 2-MBA (FIG. 49). Based on these studies,expression of ADH6 from S. cerevisiae is the most effective enzyme forthe conversion of 2-MBA to 2-MBO, though Ms-ADH1 and SFA1 were alsoeffective and likely not significantly different than the ADH6.

Maximum Production

Increasing initial glucose concentration or feeding carbon increased thefinal production of 2-MBO (FIG. 50).

GCN4 (“General Control Nondepressible”) encodes a transcriptionalactivator and was originally characterized as a positive regulator ofgenes expressed during amino acid starvation. In addition to thederepression of genes involved in 19 out of 20 amino acid biosyntheticpathways, Gcn4p may directly or indirectly regulate the expression ofgenes involved in purine biosynthesis, organelle biosynthesis,autophagy, glycogen homeostasis, and multiple stress responses.

Under environmental stresses, such as amino acid starvation, purinelimitation, or nitrogen limitation, the translation of GCN4 is induced.Gcn4p is a member of the basic leucine-zipper (bZIP) family and bindsDNA as a homodimer. Gcn4p has been shown to bind the consensus sequenceTGACTC, located upstream of many genes induced during amino acidstarvation.

Constitutive expression of GCN4 from a plasmid in the context of a GCN4+strain increased 2-MBO productivity. Strains with a chromosomal deletionin GCN4 complemented with GCN4 did not produce this phenotype.

TABLE 9 2-MBO Production of GCN4 Overexpressing Strains Strain #Description 3-MBO 2-MBO Propanol Isobutanol 8003 p415GPD GCN4 75 1369316 409 8004 p415CYC GCN4 103 2340 418 491 8031 ΔGCN4 p415CYC GCN4 10179 184 690 8032 ΔGCN4 p415GPD GCN4 91 73 340 836 8033 ΔGCN4 p415TEF GCN4105 83 247 802 8034 ΔGCN4 p415CYC GCN4 p413TEF 88 1852 264 514ILV1^(FBR) 8035 ΔGCN4 p415TEF GCN4 p413TEF 91 1763 323 718 ILV1^(FBR)

Example 16 Citramalate Pathway

The heterologous citramalate pathway (FIG. 52) provides athreonine-independent alternative to 2-oxobutanoate, an intermediate inisoleucine biosynthesis and 2-MBO. Currently, published data encompassescharacterization of this alternate pathway for isoleucine biosynthesisin Methanococcus jannaschii and Geobacter sulfurreducens.

Included here are:

-   -   1. The heterologous expression and proof of functionality of        uncharacterized putative citramalate synthase genes (annotated        as isopropylmalate synthase (leuA) from Synechocystis and T.        maritima in S. cerevisiae;    -   2. The heterologous expression of isopropylmalate dehydrogenase        (leuB) from M. jannaschii and evidence of their functionality        in S. cerevisiae;    -   3. The heterologous expression of isopropylmalate isomerase        (leuC and leuD) from M. jannaschii and evidence of their        functionality in S. cerevisiae;    -   4. The heterologous expression of the characterized cimA gene        from G. sulfurreducens. The gene was codon-optimized and shown        to be functional in S. cerevisiae;    -   5. The expression of the entire heterologous citramalate pathway        in S. cerevisiae. No published data describes the cloning and        functionality of all four genes heterologously expressed in a        single host organism; and    -   6. Deletion of carboxy-terminus portions of the leuA protein        of T. maritima to produce feedback-inhibition resistant gene        products. These D316 and L381 truncations remove a possible        allosteric domain inhibited by isoleucine.        Citramalate Synthase (cimA/leuA)

The cimA/leuA gene has been cloned from 3 sources (Synechocystis, T.maritima, and G. sulfurreducens) using either high and/or low copy yeastvectors and driven by the TEF1 or GPD constitutive promoters. To date,all three have shown some level of conversion of pyruvate and Ac-CoA tocitramalate. The Synechocystis gene driven by the TEF1 promoter producedup to 25 μM citramalate, and the G. sulfurreducens and T. maritimacimA/leuA driven by the GPD promoter yielded about a 15-fold to 50-foldincrease at 450 μM to 1600 μM, respectively (FIG. 53).

A lower level or no citramalate is detected in yeast strains withfull-length cimA/leuA when grown in SD medium supplemented with Ile(isoleucine) compared to SD medium with no Ile, suggesting a possiblepost-translational allosteric inhibition by Ile. There is a regulatorydomain on the C-terminus that, when deleted, may allow for feedbackinhibition-resistance. This truncated version has been cloned to producea feedback inhibition-resistant cimA/leuA(cimA^(FBR)/leuA^(FBR)).

Example 17 Isopropylmalate Isomerase and Isopropylmalate Dehydrogenase

Yeast express endogenous isopropylmalate isomerase (LEU1) andisopropylmalate dehydrogenase (LEU2) in the cytoplasm. When cimA/leuAvariants are introduced into an ILV1::KanMX (threonine deaminaseknock-out; isoleucine auxotroph) yeast background, complementation isdemonstrated. This suggests that LEU1 and LEU2 are sufficient for theconversion of citramalate to 2-oxobutanoate. However, the relativelylarge accumulation of citramalate (up to 1.6 mM, see above) seems toindicate that the native yeast genes are not very efficient in utilizingthe novel citramalate and erythro-β-methyl-D-malate substrates.

Therefore, three other genes were cloned and either integrated into theyeast genome or introduced as a plasmid to complete the heterologouscitramalate pathway. leuC and leuD subunits form isopropylmalateisomerase, and leuB is isopropylmalate dehydrogenase.

Data (FIG. 54) shows that introduction of the complete heterologouscitramalate pathway does increase MBO production, in particular 2-MBO.Strains containing cimA/leuA (strains E, F), leuCD and leuB (strain C),or cimA/leuA plus leuCD and leuB (strains A1, A2, B1, B2) produce MBO at1:2 or 1:3 3-MBO:2-MBO ratios without significantly increasing 3-MBOover the control. In the negative controls (strains D, G), the ratio isthe inverse at 2:1 3-MBO:2-MBO ratio.

TABLE 10 MBO Production of CimA/leuA Overexpression Strains Strain IDGenotype Media 3-MBO μM 2-MBO μM 8081 p416GPD-leuA (Tm),SD-His-Ile-Leu-Ura 176.68 149 p413TEF1-leuD (Mj) 8081 p416GPD-leuA (Tm),SD-His-Ile-Leu-Ura 136.71 214 p413TEF1-leuD (Mj) 8080 p416GPD-cimA (Gc),SD-His-Ile-Leu-Ura 186.67 112 p413TEF1-leuD (Mj) 8080 p416GPD-cimA (Gc),SD-His-Ile-Leu-Ura 147.04 313 p413TEF1-leuD (Mj) 8081 p416GPD-leuA (Tm),SD-His-Ile-Leu-Ura 117.3 261 p413TEF1-leuD (Mj) 8081 p416GPD-leuA (Tm),SD-His-Ile-Leu-Ura 119.75 364 p413TEF1-leuD (Mj) 8080 p416GPD-leuA (Gc),SD-His-Ile-Leu-Ura 131.26 232 p413TEF1-leuD (Mj) 8080 p416GPD-leuA (Gc),SD-His-Ile-Leu-Ura 162.98 561 p413TEF1-leuD (Mj) 8121 p416GPD,p413TEF1-leuD SD-His-Ile-Leu-Ura 146.26 192 (Mj) 8122 p416GPD, p413TEF1SD-His-Ile-Leu-Ura 131.69 86 8055 p416GPD-leuA (Tm), SD-His-Ile-Leu-Ura134.29 67 p415TEF1 8059 p416GPD-cimA (Gs), SD-His-Ile-Leu-Ura 128.21 107p415TEF1 8055 p416GPD-leuA (Tm), SD-Leu-Ura 94.21 209 p415TEF1 8059p416GPD-cimA (Gs), SD-Leu-Ura 113.7 228 p415TEF1 8123 p426TEF, p415TEF1SD-Leu-Ura 111.5 73 media only SD-His-Ile-Leu-Ura 0 0 8081 p416GPD-cimA(Tm), SD-His-Ile-Leu-Ura 176.68 149 p413TEF1-leuD (Mj) 8081 p416GPD-cimA(Tm), SD-His-Ile-Leu-Ura 136.71 214 p413TEF1-leuD (Mj) 8080 p416GPD-cimA(Gc), SD-His-Ile-Leu-Ura 186.67 112 p413TEF1-leuD (Mj) 8080 p416GPD-cimA(Gc), SD-His-Ile-Leu-Ura 147.04 313 p413TEF1-leuD (Mj) 8081 p416GPD-cimA(Tm), SD-His-Ile-Leu-Ura 117.3 261 p413TEF1-leuD (Mj) 8081 p416GPD-cimA(Tm), SD-His-Ile-Leu-Ura 119.75 364 p413TEF1-leuD (Mj) 8080 p416GPD-cimA(Gc), SD-His-Ile-Leu-Ura 131.26 232 p413TEF1-leuD (Mj) 8080 p416GPD-cimA(Gc), SD-His-Ile-Leu-Ura 162.98 561 p413TEF1-leuD (Mj) 8121 p416GPD,p413TEF1-leuD SD-His-Ile-Leu-Ura 146.26 192 (Mj) 8122 p416GPD, p413TEF1SD-His-Ile-Leu-Ura 131.69 86 8055 p416GPD-cimA (Tm), SD-His-Ile-Leu-Ura134.29 67 p415TEF1 8059 p416GPD-cimA (Gs), SD-His-Ile-Leu-Ura 128.21 107p415TEF1 8055 p416GPD-cimA (Tm), SD-Leu-Ura 94.21 209 p415TEF1 8059p416GPD-cimA (Gs), SD-Leu-Ura 113.7 228 p415TEF1 8123 p426TEF, p415TEF1SD-Leu-Ura 111.5 73 media only SD-His-Ile-Leu-Ura 0 0 Data normalized toOD₆₀₀ = 4.,.

There is an obvious increase in 2-MBO production when strains are grownin media +Isoleucine compared to −Isoleucine. This observation holdstrue for both strains containing the complete heterologous pathway, aswell as strains containing only cimA/leuA. The addition of cimA/leuAalone from either T. maritima or G. sulfurreducens increases 2-MBO yield2-fold, whereas introduction of the entire heterologous pathwayincreases 2-MBO up to 8-fold.

The native yeast genes BAT1 and BAT2 can work reversibly to convert Ileto 2-keto-3-methyl-valerate and 2-MBO. If this were the sole reason forthe increase of 2-MBO in the +Ile medium, we would observe acommensurate increase of 2-MBO across the board for strains A1, A2, B1,B2, C, D, E, and F. However, there is an obvious increase in 2-MBOproduction specifically in strains containing the complete citramalatepathway. This may be because when media is supplemented with Ile, thecells are under less physiological stress and can divert more of theintermediate citramalate and erythro-β-methyl-D-malate compounds to2-MBO production rather than to isoleucine biosynthesis.

Interestingly, the introduction of only leuB and leuCD (no cimA/leuA)also increases 2-MBO production about 2-fold. When only leuB and leuCare expressed, however, a functional isopropylmalate isomerase cannot beformed and MBO production is similar to that of the negative control.Although no direct functional assays have been performed on the activityand expression of the leuB and leuCD genes, this change in phenotypeimplies that these heterologous genes are functional in yeast (FIG. 55).

Example 18 Other Alternative Pathways to 2-MBO

In addition to the incorporation of the citramalate pathway (cimA) to2-oxobutanoate synthesis, other alternative pathways for 2-MBOproduction were evaluated using a stoichiometric model of yeastmetabolism that allows assessing the effects of genetic manipulations onthe maximum theoretical yields of 2-MBO from glucose. The model accountsfor the needs of the yeast cell to balance cofactors such as NAD andNADH in the pathway from glucose to 2-MBO and for the need to provideenergy in the form of ATP for the biosynthetic pathways. The model wasused to compare various alternative scenarios to wild type 2-MBOproduction, such as the effect of moving the Isoleucine (Ile) pathwayfrom mitochondrion to the cytoplasm and the utilization of a NADPHdependent glycerol-3-phosphate dehydrogenase (GAPD) instead of thenative NADH-dependent form of the enzyme. The effects of thesemanipulations on the maximum theoretical yields were assessed under bothaerobic and anaerobic conditions in order to span the range or realisticproduction environments.

The maximum theoretical 2-MBO yield from glucose for wild type (wt)yeast using the mitochondrial Ile pathway was calculated to be 0.70 molmol-1 (0.34 g g-1) aerobically and 0.29 mol mol-1 (0.14 g g-1)anaerobically (FIG. 56). Adding a cytoplasmic Ile pathway was predictedto increase the 2-MBO yield by 8.6% and 44% in aerobic and anaerobicconditions respectively. Based on these results, a cytoplasmic Ilepathway would be highly preferred especially in partially aeratedconditions. Adding the CimA pathway together with the with thecytoplasmic Ile pathway would provide an additional 2.6% and 15.4%increase in 2-MBO yield in aerobic and anaerobic conditionsrespectively. The analysis showed that combining the CimA pathway withthe native threonine-dependent 2-MBO pathway allows for more efficientbalancing of NADH and thus increases 2-MBO yields at the expense ofethanol fermentation. Expression of a NADPH dependent GAPD enzyme inyeast was predicted to result in 2.6% (aerobic) and 9.6% (anaerobic)increase in 2-MBO yield. Combining the three novel pathways (cytoplasmicIle, CimA, and NADPH-dependent GAPD) increases the maximum theoreticalyield of 2-MBO from glucose by 5.2% (aerobic) and 53.8% (anaerobic). Theoverall result of the three manipulations is predicted to be to make2-MBO production entirely independent of the level of aeration and thusprovide more flexibility for production process design.

The effect of isoleucine on LeuA activity was also examined. Strains(see table below) were grown is selective media to mid-log phase. Cellswere harvested by centrifugation and suspended in TES buffer (100 mM, pH7.5) and disrupted by bead beating. Cell debris was removed bycentrifugation (25,000×g, 30 min, 4° C.) to make cell extracts. Smallmolecules were removed by diafiltration (3×, 90% volume, 5000 D cut-offfilter). Protein concentration was determined by BCA, and finalsuspensions were diluted to 1 mg/ml.

Strain Genotype 8055 ΔILV1, p416GPD leuA(Tm)), p415TEF 8076 ΔILV1,p416GPD leuA(Tm)1-381 p415TEF 8077 ΔILV1, p416GPD leuA(Tm)1-316 p415TEFControl ΔILV1, p416TEF p415TEF

Assay mixture contained the following: sodium pyruvate, 10 mM;acetyl-CoA, tri-lithium salt, 1.0 mM; TES buffer, 100 mM at pH 7.5;DTNB, 5 mM. Reactions were initiated with the addition of dialyzed cellextract. Activity was measured by the total increase in A412 after 30minutes at 22° C., and total absorbance was subtracted from a mixturewith pyruvate excluded. Sensitivity of citramalate synthase toisoleucine, a putitive allosteric inhibitor, was tested by addingisoleucine to 10 mM (FIG. 57).

Example 19 Synthesis of bis(2-methylbutyl)ether and Use as a FuelAdditive

Oxygenated additives can be used to boost the performance of fuels.Ethers have a much lower water absorbance than alcohols and can used asa cetane enhancer. The most common way to synthesize ethers is throughthe intermolecular condensation of alcohols using an acid catalyst. Thealcohols can be 2-methylbutanol, 3-methylbutanol, a mixture of both, ora mixture of a methylbutanol and another alcohol. In the case of2-methylbutanol, the alcohol can be either the single enantiomer or aracemic mixture. The list of catalysts includes, but is not limited to,sulfuric acid, p-toluenesulfonic acid, methanesulfonic acid,trifluoromethanesulfonic acid, phosphoric acid, phosphomolybic acid,phosphotungstic acid, oxalic acid, boric acid, and hydrofluoric acid.Heterogeneous catalysts such as Nafion, acidic ion exchange catalystsand zeolites can also be used.

A preferred synthesis is:

A 5000 ml 3 neck round bottom flask, equipped with magnetic stirring,heating block, and a Dean Stark trap, was charged with2-methyl-1-butanol (2000.0 g, 22.7 mol) and triflourmethanesulfonic acid(50 ml, 0.57 mol). The solution was heated to reflux (internaltemperature≈135° C.) for 68 hours¹. During this time approximately 230ml of water had collected in the Dean Stark trap. The reaction hadturned a dark yellow. The reaction was washed with water (200 ml)followed by 1N NaOH (500 ml), and then with brine (200 ml). The liquidwas dried over Na₂SO₄ (150 g). The crude material (1500 g) was purifiedby vacuum (30 to 40 mm Hg) distillation through a 150 mm Vigreux columnto yield the product as a clear liquid (1077 g, 60% yield; GC purity: 92area %, 8% other ethers). ¹ Extended reaction times lead to degradationof the product.

This reaction was also conducted on equimolar amounts of 2-methylbutanoland 3-methylbutanol. After distillation a mixture of ethers was isolatedin 43% yield. The ratio of bis(3-methylbutyl)ether tobis(2-methylbutyl)ether to the mixed ether was 50:5:45.

Example 20 Preparation of 2-methyl-1-(2-methylbutoxy)butane

This example demonstrates the conversion of 2-methylbutyl ether to2-methyl-1-(2-methylbutoxy)butane, otherwise known asbis-(2-methylbutyl)ether from 2-methylbutanol.

A 1000 mL 3-neck round bottom flask equipped with magnetic stirring,heating mantle, and a Dean-Stark trap was charged with2-methyl-1-butanol (400.0 g, 4.54 mol) and trifluoromethanesulfonic acid(10 mL, 0.11 mol). The use of triflic acid was found to provide superiorresults compared to other catalysts. The solution was heated to refluxfor 56 to 72 hours. During this time, approximately 43 mL of water hadcollected in the Dean-Stark trap. The reaction turned either pale yellowor black depending on the reaction time. The reaction was washed with 1NNaOH (100 mL), then saturated NaHCO₃ (50 mL) and finally with brine (25mL). The liquid was dried over Na₂SO₄. The crude material was purifiedby vacuum distillation (ca. 50 mmHg, b.p. 52 to 72° C.).

A reaction time of 56 hours gave a 70% yield of product. A reaction timeof 70 hours gave a 35% yield of product. The purity of the product wasanalyzed by gas chromatography (GC). In the procedure wherein thereaction time was 56 hours, the GC peak area of a sample of the reactionmixture reflected the following amounts: 74% desired product, 18%alcohol, and 8% other ethers. In the procedure wherein the reaction timewas 70 hours, the GC peak area of a sample of the reaction mixturereflected the following amounts: 98% desired product, 2% other ethers.

In addition, the bis-(2-methylbutyl)ether was tested as a diesel fueladditive, and was found to have a calculated cetane number between 126and 160.

Example 21 Use of MBO in the Production of Biodiesels

Biodiesel, an alternative diesel fuel derived from vegetable oil, animalfats, or waste vegetable oils, is obtained by the transesterification oftriglycerides with an alcohol in presence of a catalyst to give thecorresponding alkyl esters. It provides a market for excess productionof vegetables oils and animal fats; it decreases the country'sdependence on imported petroleum; it is a renewable fuel and does notcontribute to global warming due to its closed carbon cycle; it haslower exhaust emissions than regular diesel fuel; and can be used indiesel engines without extensive engine modifications.

Two major indicators of biodiesel fuel quality are: the cloud point, thetemperature at which waxy solids first appear during the cooling ofdiesel fuel; and the cetane number (CN), the measure of the readinessfuel to autoignite when injected into the engine.

This example shows the synthesis of biodiesel 2-methylbutyl esters andbiodiesel methyl esters, compares the cloud points and cetane numbers,and demonstrates that methylbutyl esters are a viable replacement formethyl esters.

Soybean oil 2-methylbutyl esters and canola oil 2-methyl butyl esterswere synthesized by transesterifying the triglyceride with 2-methylbutylalcohol in presence of catalyst. The catalyst could be acidic or basic,aqueous or organic, free or bounded on solid support; it includes but isnot limited to: potassium hydroxide, sodium hydroxide, sulfuric acid,p-toluenesulfonic acid, potassium carbonate, sodium hydride, DOWEXMarathon A OH form, magnesium oxide, and calcium oxide.

Synthesis of 2-methylbutyl esters. A solution of canola oil (530.00 g)in 2-methylbutanol (370.79 g, 4.206 mol) was stirred at 50° C.; sulfuricacid (0.61 g, 0.006 mol, 0.01 equiv.) was added. The reaction mixturecontinued stirring at reflux (˜125° C.) until diglycerides were notdetected by GC-FID (˜70 h). The reaction mixture was allowed to cool toambient temperatures and transferred to a separatory funnel whereglycerin was allowed to settle and then removed. The remaining solutionwas washed with sat'd aq. NaHCO₃ (500 mL), sat'd aq. NHCl₃ (2×500 mL),brine (500 mL), and dried over Na₂SO₄ (100.00 g). The crude product wasdistilled under vacuum to afford 2-methylbutyl esters as an off-whiteliquid. Yield 573.76 g.

Soybean oil methyl esters and canola oil methyl esters were synthesizedby transesterifying the triglyceride with methanol in presence ofcatalyst. The catalyst could be acidic or basic, aqueous or organic,free or bounded on solid support; it includes but is not limited to:potassium hydroxide and sodium hydroxide.

Synthesis of methyl esters. Canola oil (400.20 g) was stirred at 60° C.;potassium hydroxide (1.80 g, 0.028 mol) dissolved in methanol (58.12 g,1.814 mol) was added. The reaction mixture continued stirring at reflux(˜63° C.) until triglycerides were not detected and diglycerides levelswere low by GC-FID (˜5 h). The reaction mixture was allowed to cool toambient temperatures and transferred to a separatory funnel whereglycerin was allowed to settle and be removed. The remaining solutionwas washed with sat'd aq. NHC13 (2×250 mL), brine (2×200 mL), and driedover Na2SO4 (40.00 g). Crude yield 396.19 g. The crude product wascombined with a second lot of canola oil methyl esters and distilledunder vacuum to afford methyl esters as an off-white liquid.

Biodiesel Fuel Properties for Methyl Esters and 2-Methylbutyl EstersTest Method Methy Esters 2-Methylbutyl Esters Triglyceride — Soybean oilCanola oil Soybean oil Canola oil Cloud Point, ° C. ASTM D2500 −5 −6 −7−11 Total Glycerin, ASTM D6584 0.169 0.172 0.139 0.142 % mass FreeGlycerin, % ASTM D6584 0.006 0.006 0.008 0.007 mass Cetane Number, B20ASTM D613 43.7 41.7 41.1 43.4 Cetane Number Extrapolated 51.7 41.7 38.750.2 Notebook Ref. — 1053-82-1 1053-83-1 1077-73E 1077-74F

The table above shows the fuel properties of soybean 2-methylbutylesters, canola 2-methylbutyl esters, soybean methyl esters and canolamethyl esters that. The cloud points and the cetane numbers of2-methylbutyl esters and the methyl ester were comparable.

Example 22 Differentiation of Biological MBO and Ethers Thereof

A chirality test is used to determine the source of the MBO. MBOproduced biologically using the methods of the present invention will bechiral, but chemically produced MBO will be racemic. 2-MBO enantiomerswere successfully separated on the BGB-174 chiral column (FIGS. 58A and58B). This demonstrated that the samples are authentic and contain onlyS-2-MBO. Three various derivatives were generated independently for thistest: the trimethyl-silyl, the trifluouroacetate, and the benzoatederivatives of 2-MBO.

The R- and S-2 MBO racemic mixture was separated using GCMS under thefollowing experimental conditions:

-   -   Column: BGB-174, 30 m×250 μm×0.25 μm, BGB-Analytik P/N 27430-025    -   Carrier gas: helium, 17 psi.    -   Oven: 60° C. for 5 min, then 0.5° C./min to 70° C. for 0.5 min.    -   Injector: split 1:40, temp 220° C., 1 μL injection volume.    -   Detector: MS, SIM ion of 57.1.    -   Samples prepared in CH₂Cl₂ at approximately 1 mM.    -   S-2-MBO was extracted from the fermentation broth with methylene        chloride under conditions similar to those presented above (FIG.        58C).

Example 23 Exemplary YAC Constructs

The following gene combinations were used to assemble yeast artificialchromosomes encoding specific enzymes in the pathways of interest forthe production of 2-MBO. See FIGS. 59-73 for schematic illustrations ofthe assemblies.

GENE YAC6* YAC8* YAC10* YAC7* YAC9* YAC5* YAC5Δ* YAC14* YAC14Δ* MDH2 X X(Ps) PYC1 X X X X X X (Ps) AAT2 X X X X X X (Ps) HOM3^(FBR) X X X* X* X*X* (Ps) HOM2 X X X X X X (Ps) HOM6 X X X X X X (Ps) THR1 X X X X X X(Ps) THR4 X X X X X X (Ps) ILV1 X* (Ps)^(FBR) ILV1 (Ps) X* X* X* Δ15IlvA (Ec) X ILV2 (Ps) X X ILV2 X X (Ps) Δ26 ILV6 (Ps) X X X X ilvG (Ec)X ILV5 (Ps) X X ILV5 (Ps) X X Δ40 IlvC (Ec) X ILV3 (Sc) ILV3 (Ps) X XILV3 X X (Ps) Δ34 IlvD (Ec) X PDC 3-6 X X X (Ps) KdcA (Ll) X X ADH6 X XX X X (Sc) X* = X(^(p)TEF & ^(p)CUP) (Ps) = Pichia stipitis version (Ec)= Escherichia coli version (Sc) = Saccharomyces cerevisiae version (Ll)= Lactococcus lactis version

Example 24 Methods of Recovery of MBO and Other Compounds Produced inCulture Media and Analysis by GC-FID

GC-FID was used to monitor a variety of produced compounds includingmethanol, ethanol, n-propanol, n-butanol, i-butanol, sum of isovalericacid−2MeBu acid, 2-MBO, 3-MBO, and 2,3-butanediol.

Instrument and analysis conditions:

-   -   Agilent 7890A gas chromatography system.    -   Flow: 1.1 ml/min He (39.5 cm/sec 45° C.), constant flow control.    -   Gradient Timetable: 45° C. for 3.2 min, then 25° C./min to 60°        C., then 45° C./min to 200° C. Post run time is at 230° C. for        2.2 min.    -   Column: DB-624 (Agilent), 20 m×0.18 mm×1 μm.    -   Inlet: 230° C., split 40:1 (back) 100:1 (front).    -   Detector: FID at 230° C., H₂ 45 ml/min, air 450 ml/min, Constant        column+makeup gas 50 ml/min.    -   Needle rinse: 1—acetonitrile, 2—water.    -   Injection type: simultaneous injection into two identical        columns of two different samples or two identical standard        working solutions.    -   Injection volume: 1 μl.    -   Quantification: Internal standard method (IS: 1-pentanol).        Calibration is set between 50 mM-25 μM for ethanol, 5 mM-25 μM        for n-propanol, i-butanol, and n-butanol, 10 mM-50 μM for        methanol, and 1 mM-5 μM for MBO and MBO acid.

Representative chromatograms showing the separation of compounds areprovided in FIGS. 74 (a-c). Other methods of recovery include gasstripping, fractional distillation, chromatography, pervaporation,adsorption, and solid-liquid extraction.

Example 25 GC/MS Analysis of MBO-Related Compounds Extracted in CH₂Cl₂

MBO and derivatives found in spent media were extracted in CH₂Cl₂ andanalyzed by GC/MS. The internal standard method was applied forquantification (IS 1-pentanol). No pH adjustment was needed. Samplepreparation: add 900 μl methylene chloride, 200 μl of 1-pentanol inNa₂SO₄ aqueous solution, and 500 μl sample or standard in a GC vial.Vortex the vial, allow 5 min for the phases to separate, and analyze theorganic phase without removing the aqueous phase. The LOQ is 3 μM (seerepresentative chromatograms in FIG. 75 (a-c)).

Instrument and Analysis Conditions:

-   -   Agilent 7890A gas chromatography system connected to a 5975C        inert MSD mass spectrometer. The method is called “MBOorg3”.    -   Flow: 0.75 ml/min He, constant flow control.    -   Gradient Timetable: 70° C. for 1.0 min, then 10° C./min to        110° C. for 0.5 min, then 20° C./min to 140° C. for 0.5 min.        Post run time is at 200° C. for 2.0 min.    -   Column: Rtx624 (Restek), 20 m×0.18 mm×1 um.    -   Inlet: 230° C., split 20:1.

Detector: MS with interface temperature set to 230° C. The selective ionmonitoring is set for ions 55, 57, 58, and 70. The scan analysis wasalso run simultaneously for the mass range 35-200 m.u.

-   -   Masses for SIM analysis (dwell time 3 msec).

2-Me-butyraldehyde 57.1 Isovaleraldehyde 58.1 2-Me-butanol 55.13-Me-butanol 57.1 2-Me-butyl acetate 70.1 Isopentyl acetate 70.1

-   -   Needle rinse: 1—methylene chloride:methanol 1:1, 2—methylene        chloride    -   To avoid carry-over, the syringe is washed three times with        solvent 1, then solvent 2, and eight times with the sample.    -   Injection volume: 1 μl.    -   Quantification: Internal standard method (IS: 1-pentanol).        Calibration is set between 10 mM-3 μM for the alcohols and        aldehydes, and 5 mM-1.5 μM for the esters.

Example 26 MBO and Ether Fuel Properties

Alcohol Fuels Property Ethanol Butanol 2-MBO 3-MBO Gasoline ChemicalFormula C₂H₆O C₄H₁₀O C₅H₁₂O C₅H₁₂O C₄-C₁₂ Properties Oxygen (% wt) 35   22    18    18 0 Physical Flash Point, min (° C.) 13    36    43   43 −43 Properties Boiling Point, max (° C.) 79    118    130    130 27-225 Freezing Point, max (° C.) −114    −90    −70   −117 −40 FuelSolubility in Water (wt %)¹ 100     8     3     2 Immiscible WaterSolubility in Fuel (wt %)¹ 100    21     9     9 Immiscible FuelProperties - Energy Content (BTU/gal) 84,519 107,196 113,627 112,240109,000-119,000 Neat Fuel Pump Octane (R + M)/2² 130.9    105.7    102.0   101.0 80-85 Reid Vapor Pressure (psi) 2.3     0.7     2.3     2.37.8-15  Fuel Properties - Energy Content (BTU/gal) 111,052 113,320113,963 113,824 NA 10% Blend in Pump Octane (R + M)/2 85.9    83.3   83.0    82.0 Gasoline³ Reid Vapor Pressure (psi) ~8.2^(A)   ~7^(B)  ~7^(B)   ~7^(B) Vol % to 2.7 wt % O₂ ⁴ 7.8    12.5    14.8    14.8Comparison of petroleum gasoline with substitutable alcohol fuels ¹Basedon literature review ²Extrapolated from tests on blended fuels (i.e.these are blending octanes) ³The testing was performed with an RBOBtested at a Pump Octane of 80.85 and an RVP of 7.37 ⁴Oxygen contentrequirement in US Reformulated Gasoline markets ^(A)RVP was measured at8.8 psi for 5% blend in RBOB; reported value for 10% blend is based onliterature review ^(B)Value was measured with 9.5% volumetric blend ofbutanol (or 2MBO) in RBOB

There are considerations in determining blend levels for 2-MBO and 3-MBOin gasoline. Clean air regulations in the US have historically requiredthe presence of oxygen in gasoline in amounts between 2% and 2.7% (inweight). Given that 18% of MBO in weight is oxygen (while conventionalgasoline contains no oxygen) and making adjustment for the differentdensity of MBO and gasoline, then in order to satisfy the 2-2.7% range,you can add between 9.5-13% of MBO in gasoline (the number for ethanolis only 5-7% because ethanol has higher oxygen weight %). Similarcalculations can be made for any similar restrictions in oxygen content.

Fuel Properties: MBO & Other Alcohol Fuels vs. Regular Gasoline MeasuredImplied Measured Implied RON¹ MON² Octane Octane RVP³ RVP Fuel testedDescription N^(o) N^(o) N^(o) N^(o) psi psi Considerations RBOBReference 78.9 82.8 80.9 — 7.4 — for octane & RVP testing of alcoholfuels 5% EtOH/ ~2% 81.5 85.2 83.4 130.9 8.8 35.6  RVP for EtOH 95% RBOBoxygen blends is not level linear 8% 1- ~2% 80.8 84.9 82.9 105.9 7.1 4.4Butanol/ oxygen 92% RBOB level 8% ~2% 82.0 86.0 84.0 120.2 7.1 4.5isoButanol/ oxygen 92% level RBOB 11% ~2.7% 82.4 87.4 84.9 117.7 7.0 4.4isoButanol/ oxygen 89% level RBOB 9.5% ~2% 81.0 85.0 83.0 103.5 7.0 3.72MBO/ oxygen 90.5% level RBOB 13% 2MBO/ ~2.7% 81.4 85.7 83.6 101.6 6.93.7 87% oxygen RBOB level

Example 27 Ether Fuel Testing

Solubility Measured Implied in Water Cetane³ Cetane³ Pour Cloud CFPP¹Flash Lubricity⁴ Fuel tested ppm N^(o) N^(o) ° C. ° C. ° C. ° F. cpULSD² Reference fuel for 45.7 — −18 −13 −16 176 340 testing ULSD w/2%3MBO-ether 46.3 75.7 −18 −13 −16 173 ULSD w/10% 3MBO-ether 48.3 71.7ULSD w/2% 2MBO-ether 48.0 160.7 ULSD w/10% 2MBO-ether 53.8 126.7 ULSDw/10% 2MBO-ether 152 ULSD w/10% 3MBO-ether 162 2MBO-ether <500 118Hexadecane Reference for 534 lubricity testing Hexadecane w/2% 3MBO-<−24 <−21 <−50 610 ether ¹Cold Filter Plugging Point ²Ultra Low SulfurDiesel ³ASTM Requirements for Cetane: >40 ⁴ASTM Requirements for FlashPoint: >100° F.

REFERENCES

-   Rieder, S. E., and Emr, S. C. (2000). Overview of subcellular    fractionation procedures for yeast Saccharomyces cerevisiae. Current    Protocols in Cell Biology: 3.7.1-3.7.25.-   Rieder, S. E., and Emr, S. C. (2000). Isolation of subcellular    fractions from yeast Saccharomyces cerevisiae. Current Protocols in    Cell Biology: 3.8.1-3.8.68.-   Gocke D, Nguyen C L, Pohl M, Stillger T., Walter L, Iler M. M.    (2007). Branched-Chain Keto Acid Decarboxylase from Lactococcus    lactis (KdcA), a Valuable Thiamine Diphosphate-Dependent Enzyme for    Asymmetric C—C Bond Formation Adv. Synth. Catal. 349: 1425-1435.-   Berthold C. L., Gocke D., Wood M. D., Leeper F. J., Pohl M.,    Schneider G. (2007). Structure of the branched-chain keto acid    decarboxylase (KdcA) from Lactococcus lactis provides insights into    the structural basis for the chemoselective and enantioselective    carboligation reaction. Acta Crystallographica Section D. 63:    1217-1224.

What is claimed is:
 1. A recombinant microorganism comprising at leastone exogenous nucleic acid molecule, wherein the at least one exogenousnucleic acid molecule encodes (i) a pyruvate decarboxylase having atleast 90% amino acid sequence identity to a Pichia stipitis pyruvatedecarboxylase PDC3-6 gene product of SEQ ID NO:53 having pyruvatedecarboxylase activity and (ii) an alcohol dehydrogenase that catalyzesa 2-methylbutanal to 2-methylbutanol conversion; and wherein therecombinant microorganism produces 2-methylbutanol.
 2. The recombinantmicroorganism of claim 1, wherein the alcohol dehydrogenase is from agenus selected from the group consisting of Saccharomyces, Pichia, orMycobacterium.
 3. The recombinant microorganism of claim 2, wherein thealcohol dehydrogenase has at least 90% amino acid sequence identity toSaccharomyces cerevisiae ADH6 gene product of SEQ ID NO:66 havingalcohol dehydrogenase activity.
 4. The recombinant microorganism ofclaim 2, wherein the alcohol dehydrogenase has at least 90% amino acidsequence identity to Saccharomyces cerevisiae SFA1 gene product of SEQID NO:76 having alcohol dehydrogenase activity.
 5. The recombinantmicroorganism of claim 1, wherein the recombinant microorganism is froma genus selected from the group consisting of Escherichia, Pseudomonas,Bacillus, Corynebacterium, Clostridium, Lactobacillus, Pichia, orSaccharomyces.
 6. A recombinant microorganism comprising at least oneexogenous nucleic acid molecule, wherein the at least one exogenousnucleic acid molecule encodes (i) a pyruvate decarboxylase having atleast 90% amino acid sequence identity to a Pichia stipitis pyruvatedecarboxylase PDC3-6 gene product of SEQ ID NO:53 having pyruvatedecarboxylase activity, wherein said recombinant microorganism produces2-methylbutanol.
 7. The recombinant microorganism of claim 6, furthercomprising at least one exogenous nucleic acid molecule encoding analcohol dehydrogenase that catalyzes a 2-methylbutanal to2-methylbutanol conversion.
 8. The recombinant microorganism of claim 7,wherein the alcohol dehydrogenase is from a genus selected from thegroup consisting of Saccharomyces, Pichia, or Mycobacterium.
 9. Therecombinant microorganism of claim 8, wherein the alcohol dehydrogenasehas at least 90% amino acid sequence identity to Saccharomycescerevisiae ADH6 of SEQ ID NO:66 having alcohol dehydrogenase activity.10. The recombinant microorganism of claim 8, wherein the alcoholdehydrogenase has at least 90% amino acid sequence identity toSaccharomyces cerevisiae SFA1 of SEQ ID NO:76 having alcoholdehydrogenase activity.
 11. A recombinant method for producing a2-methylbutanol, comprising culturing the recombinant microorganism ofclaim 1 in a culture medium containing a carbon source, wherein therecombinant microorganism produces spent culture medium from the culturemedium by metabolizing the carbon source to the 2-methylbutanol; andrecovering the 2-methylbutanol from the spent culture medium.
 12. Themethod of claim 11, wherein the alcohol dehydrogenase is from a genusselected from the group consisting of Saccharomyces, Pichia, orMycobacterium.
 13. The method of claim 12, wherein the alcoholdehydrogenase has at least 90% amino acid sequence identity toSaccharomyces cerevisiae ADH6 of SEQ ID NO:66 having alcoholdehydrogenase activity.
 14. The method of claim 12, wherein the alcoholdehydrogenase has at least 90% amino acid sequence identity toSaccharomyces cerevisiae SFA1 of SEQ ID NO:76 having alcoholdehydrogenase activity.
 15. The method of claim 11, wherein therecombinant microorganism is selected from a genus selected from thegroup consisting of Escherichia, Pseudomonas, Bacillus, Corynebacterium,Clostridium, Lactobacillus, Pichia, or Saccharomyces.
 16. The method ofclaim 15, wherein the recombinant microorganism is Escherichia coli. 17.The method of claim 15, wherein the recombinant microorganism isSaccharomyces cerevisiae.
 18. The method of claim 15, wherein therecombinant microorganism is Pichia stipitis.
 19. A recombinant methodfor producing a 2-methylbutanol, comprising culturing the recombinantmicroorganism of claim 6 in a culture medium containing a carbon source,wherein the recombinant microorganism produces spent culture medium fromthe culture medium by metabolizing the carbon source to the2-methylbutanol; and recovering the 2-methylbutanol from the spentculture medium.
 20. A recombinant method for producing a2-methylbutanol, comprising culturing the recombinant microorganism ofclaim 7 in a culture medium containing a carbon source, wherein therecombinant microorganism produces spent culture medium from the culturemedium by metabolizing the carbon source to the 2-methylbutanol; andrecovering the 2-methylbutanol from the spent culture medium.